Aspects of programmed cell death during early senescence of barley leaves: possible role of nitric oxide

Summary. Leaf senescence is a highly coordinated process which involves programmed cell death (PCD). Early stages of leaf senescence occurring during normal leaf ontogenesis, but not triggered by stress factors, are less well known. In this study, we correlated condensation of chromatin and nuclear DNA (nDNA) fragmentation, two main features of PCD during early senescence in barley leaves, with the appearance of nitric oxide (NO) within leaf tissue. With the help of the alkaline version of the comet assay, together with measurements of nDNA fluorescence intensity, we performed a detailed analysis of the degree of nDNA fragmentation. We localised NO in vivo and in situ within the leaf and photometrically measured its concentration with the NO-specific fluorochrome 4-amino-5-methylamino-2′,7′-difluorofluorescein. We found that both nDNA fragmentation and chromatin condensation occurred quite early during barley leaf senescence and always in the same order: first nDNA fragmentation, in leaves of 6-day-old seedlings, and later chromatin condensation, in the apical part of leaves from 10-day-old seedlings. PCD did not start simultaneously even in neighbouring cells and probably did not proceed at the same rate. NO was localised in vivo and in situ within the cytoplasm, mainly in mitochondria, in leaves at the same stage as those in which chromatin condensation was observed. Localisation of NO in vascular tissue and in a large number of mesophyll cells during the senescence process might imply its transport to other parts of the leaf and its involvement in signalling between cells. The fact that the highest concentration of NO was found in the cytoplasm of mesophyll cells in the earliest stage of senescence and lower concentrations were found during later stages might suggest that NO plays an inductive role in PCD. Correspondence: A. Mostowska, Department of Plant Anatomy and Cytology, Institute of Experimental Biology of Plants, University of Warsaw, Miecznikowa 1, 02-096 Warsaw, Poland.     

DNA fragmentation in cereal roots indicative of programmed root cortical cell death

In cereals, a progressively increasing root cortical cell death (RCD) occurs from the root tip and upwards when measured with vital staining methods. In this study, nuclear DNA fragmentation was studied in seminal root segments of wheat and barley in order to investigate if the cell death resembled apoptosis. The fraction of cells with TUNEL-positive nuclei increased gradually with increasing root age in both the cortex and the stele. Southern analysis showed a typical ladder pattern, indicating nucleosomal fragmentation already in 2-day-old root segments, and this became more pronounced in older root segments. DNA fragmentation appeared to be more extensive in wheat than in barley roots. These results confirm earlier studies, where RCD has been found to be earlier initiated and to proceed at a faster rate in wheat. The characteristic DNA fragmentation found in the roots indicates programmed cell death with mechanistic similarities to apoptosis. Ultrastructural examination of nuclei in cortex cells with transmission electron microscopy revealed an increased chromatin condensation in older roots, particularly in wheat.
In addition, we found nucleosomal DNA ladders in young leaf tissue from wheat but not from barley.     

Chemical-induced apoptotic cell death in tomato cells: involvement of caspase-like proteases

 A new system to study programmed cell death in plants is described. Tomato (Lycopersicon esculentum Mill.) suspension cells were induced to undergo programmed cell death by treatment with known inducers of apoptosis in mammalian cells. This chemical-induced cell death was accompanied by the characteristic features of apoptosis in animal cells, such as typical changes in nuclear morphology, the fragmentation of the nucleus and DNA fragmentation. In search of processes involved in plant apoptotic cell death, specific enzyme inhibitors were tested for cell-death-inhibiting activity. Our results showed that proteolysis plays a crucial role in apoptosis in plants. Furthermore, caspase-specific peptide inhibitors were found to be potent inhibitors of the chemical-induced cell death in tomato cells, indicating that, as in animal systems, caspase-like proteases are involved in the apoptotic cell death pathway in plants. Received: 5 August 1999 / Accepted: 14 March 2000     

Earlier onset of DNA fragmentation in leaves of wheat compared to barley and rye

We have found extensive nucleosomal fragmentation of native DNA extracted from leaves of healthy cereal plants, as indicated by ladder patterns on agarose gels and TUNEL staining. The time of first appearance of fragmentation differed among cereals. Native DNA from the first leaf of 10-day-old plants formed a clear ladder pattern of multiples of 180 bp fragments in wheat and triticale but not in barley and oats. In one cultivar of rye a weak ladder pattern occurred but not in another. Freezing and thawing of samples before DNA extraction resulted in much more extensive DNA fragmentation in wheat but not in rye and barley, indicating that DNA-degrading enzymes are present in the cytoplasm of wheat, but not in barley and rye, at this stage. In barley, nucleosomal fragmentation was first detected in 25-day-old plants. These results indicate that programmed cell death takes place in developing leaves of young cereal plants, but that the time of onset differs among cereal species.     

Aspects of programmed cell death during leaf senescence of mono- and dicotyledonous plants

Summary Leaf senescence is a highly regulated stage in the plant life cycle, leading to cell death, recently examined as a type of the programmed cell death (PCD). One of the basic features of PCD is the condensation of nuclear chromatin which is caused by endonucleolytic degradation of nuclear DNA (nDNA). In our investigations, we applied the technique of the single-cell electrophoresis system (“comet assay”) in order to determine the type of nDNA fragmentation during leaf senescence. The comet assay, a sensitive method revealing nonrandom internucleosomal damage that is specific for PCD, is especially useful for the detection of nDNA degradation in isolated viable cells. Simultaneously, we analyzed the mesophyll cell ultrastructure and the photosynthetic-pigment concentration in the leaves of two species,Ornithogalum virens andNicotiana tabacum, representing mono- and dicotyledonous plants which differ in the pattern of leaf differentiation. These investigations demonstrated that, in both species, the comet assay revealed nDNA degradation in yellow-leaf protoplasts containing chloroplasts that showed already changed ultrastructure (swelled or completely degraded thylakoids) and cell nuclei with a significant condensation of chromatin. There was no nDNA degradation in green-leaf protoplasts containing differentiated chloroplasts with numerous grana stacks and nuclei with dispersed chromatin. The analysis of intermediate developmental stage showed that the degradation of nDNA precedes condensation of nuclear chromatin. Thus the comet assay is a very useful and sensitive method for early detection of PCD. Moreover, results of our studies indicate that leaf senescence involves PCD.     

Senescence and programmed cell death: substance or semantics?

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.     

Spatial and temporal regulation of DNA fragmentation in the aleurone of germinating barley

During germination of barley grains, the appearance of DNA fragmentation started in aleurone cells near the embryo and extended to the distal end in a time-dependent manner. DNA fragmentation was demonstrated to occur only after the expression of -amylase mRNA in the aleurone layer. In addition, cell wall degradation started in cells near the embryo on the sides facing the endosperm. Subsequently cell wall degradation extended to the lateral cell walls and to cells more to the distal end of the grain. A typical alteration of the nucleus was observed by electron microscopy and an almost complete degradation of DNA was found in the nucleus while the nuclear envelope remained intact. The results indicate that programmed cell death occurred in aleurone cells during germination. A model is proposed for the regulation of programmed cell death in aleurone cells during germination involving ABA levels and cell wall degradation.     

Programmed cell death of secretory cavity cells in fruits of Citrus grandis cv. Tomentosa is associated with activation of caspase 3-like protease

Previously, we found that secretory cell degradation typically occurred through programmed cell death during secretory cavity development in Citrus sinensis L. (Osbeck). This finding indicated that secretory cavities could be utilized as a new cell biology model for investigating the regulatory mechanisms of plant programmed cell death. To study further the programmed cell death during secretory cavity development in Citrus fruit, we studied the morphogenetic characteristics of secretory cavities during their development in Citrus grandis cv. Tomentosa. Using light microscope- and electron microscope-TUNEL assays, immunohistochemistry and immunocytochemistry, we described the precise spatial and temporal alterations in caspase 3-like distribution, chromatin condensation and DNA fragmentation during the programmed cell death of secretory cavity cells. Caspase 3-like was found to be significantly located in both the cytoplasm and the nucleus of secretory cavity cells undergoing programmed cell death, and caspase 3-like is closely associated with chromatin condensation and DNA fragmentation. Interestingly, both caspase 3-like and DNA fragmentation were detected in the nucleoli. Our findings suggest that caspase 3-like may be involved in the programmed cell death of secretory cavity cells, especially in chromatin condensation, DNA fragmentation, nuclear degradation and the degradation of certain organelles.     

Programmed cell death during pollination-induced petal senescence in petunia

Petal senescence, one type of programmed cell death (PCD) in plants, is a genetically controlled sequence of events comprising its final developmental stage. We characterized the pollination-induced petal senescence process in Petunia inflata using a number of cell performance markers, including fresh/dry weight, protein amount, RNA amount, RNase activity, and cellular membrane leakage. Membrane disruption and DNA fragmentation with preferential oligonucleosomal cleavage, events characteristic of PCD, were found to be present in the advanced stage of petal senescence, indicating that plant and animal cell death phenomena share one of the molecular events in the execution phase. As in apoptosis in animals, both single-stranded DNase and double-stranded DNase activities are induced during petal cell death and are enhanced by Ca(2+). In contrast, the release of cytochrome c from mitochondria, one commitment step in signaling of apoptosis in animal cells, was found to be dispensable in petal cell death. Some components of the signal transduction pathway for PCD in plants are likely to differ from those in animal cells.     

Is internucleosomal DNA fragmentation an indicator of programmed death in plant cells?

Specific DNA fragmentation into oligonucleosomal units occurs during programmed cell death (PCD) in both animal and plant cells, usually being regarded as an indicator of its apoptotic character. This internucleosomal DNA fragmentation is demonstrated in tobacco suspension and leaf cells, which were killed immediately by freezing in liquid nitrogen, and homogenization or treatment with Triton X-100. Although these cells could not activate and realize the respective enzymatic processes in a programmed manner, the character of DNA fragmentation was similar to that in the cells undergoing typical gradual PCD induced by 50 microM CdSO4. This internucleosomal DNA fragmentation was connected with the action of cysteine proteases and the loss of membrane, in particular tonoplast, integrity. The mechanisms of DNase activation in the rapidly killed cells, hypothetical biological relevance, and implications for the classification of cell death are discussed.     

DNA degradation in the leaves of the sugar beet (Beta vulgaris L.) in the process of senescence

Programmed cell death (PCD) is a characteristic of all living beings in process of their development and in response to biotic and abiotic factors effects. On the pattern of the growing old 7-8th leaves of the sugar-beet (Beta vulgaris L.) of the first year vegetation of Belocerkovskaya singl-seed-45 sort character of the degradation and state of the methylation GTCGAC- and CTGCAG-sequences of the DNA were investigated. It was established by a method of electrophoresis that in the growing old leaves which didn't have any external indications of the senescence yet the DNA fragmentation didn't have place. The degradation of the total and nuclear DNA became intensive as far as senescence was gradually increasing with the age. The DNA degradation is accompanied by high level of its methylation in recognition sites of Pst I and Sal I which doesn't change during senscence of the leaves.     

Molecular analysis of programmed cell death during senescence in Arabidopsis thaliana and Brassica oleracea: cloning broccoli LSD1, Bax inhibitor and serine palmitoyltransferase homologues

This study was undertaken to characterize the programmed cell death (PCD) processes that occur during detached and natural on-plant senescence and correlate them with the expression of putative regulatory genes that may be involved in the process. DNA fragmentation and TUNEL analysis of broccoli florets showed that DNA was processed into fragments of approximately 180 bp after 48 h of harvest-induced tissue senescence. Characteristic laddering patterns were also visible in Arabidopsis leaves undergoing natural on-plant senescence and during detached senescence. Several recently isolated plant proteins have been assigned a PCD role, for example, the zinc finger containing protein, LSD1 (lesion simulating disease); Bax inhibitor (BI); and serine palmitoyltransferase (SPT), an enzyme in the sphingolipid signalling pathway. Two cDNAs encoding each of these proteins were isolated from broccoli (BoBI-1, BoBI-2, BoLSD1, BoLSD2, BoSPT1, BoSPT2), and the mRNAs increased during harvest-induced senescence in floret tissue. Expression of the Arabidopsis homologues (AtBI-1, AtLSD1, AtSPT1) were also characterized during detached leaf senescence in Arabidopsis leaves. AtBI-1 expression was constitutively expressed during detached senescence, AtLSD1 expression remained constitutively low, and AtSPT1 expression increased during detached senescence.     

Ultrastructure of Mesophyll Cells and Pigment Content in Senescing Leaves of Maize and Barley

Leaf senescence is a genetically regulated stage in the plant life cycle leading to death. Ultrastructural analysis of a particular region of the leaf and even of a particular mesophyll cell can give a clear picture of the time development of the process. In this study we found relations between changes in mesophyll cell ultrastructure and pigment concentration in every region of the leaf during leaf senescence in maize and barley. Our observations demonstrated that each mesophyll cell undergoes a similar senescence sequence of events: a) chromatin condensation, b) degradation of thylakoid membranes and an increase in the number of plastoglobules, c) damage to internal mitochondrial membrane and chloroplast destruction. Degradation of chloroplast structure is not fully correlated with changes in photosynthetic pigment content; chlorophyll and carotenoid content remained at a rather high level in the final stage of chloroplast destruction. We also compared the dynamics of leaf senescence between maize and barley. We showed that changes to the mesophyll cells do not occur at the same time in different parts of the leaf. The senescence damage begins at the base and moves to the top of the leaf. The dynamics of mesophyll cell senescence is different in leaves of both analyzed plant species; in the initial stages, the process was faster in barley whereas in the later stages the process occurred more quickly in maize. At the final stage, the oldest barley mesophyll cells were more damaged than maize cells of the same age.     

Senescence mechanisms

Senescence in plants is usually viewed as an internally programmed degeneration leading to death. It is a developmental process that occurs in many different tissues and serves different purposes. Generally, apoptosis refers to programmed death of small numbers of animal cells, and it shows some special features at the cell level. Some senescing plant cells show some symptoms typical of apoptosis, while others do not. This review will focus primarily on leaf senescence with ultimate aim of explaining whole plant senescence (i.e., monocarpic senescence). Traditionally, the ideas on senescence mechanisms fall into two major groupings, nutrient deficiencies (e.g., starvation) and genetic programming (i.e., senescence-promoting and senescence-inhibiting genes). Considerable evidence indicates that nutrient deficiencies are not central senescence program components, while increasing evidence supports genetic programming. Because chlorophyll (Chl) and chloroplast (CP) breakdown are so prominent, leaf senescence is generally measured in terms of Chl loss. Although CP breakdown may not be the proximate cause of leaf cell death, it certainly is important as a source of nutrients for use elsewhere, e.g., for developing reproductive structures in monocarpic plants, and this loss limits assimilatory capacity. The CP is dismantled in an orderly sequence. Individual protein complexes seem to be taken out all at once, not one subunit at a time. Removal of any component, e.g., Chl, seems to destabilize the whole complex. It is of special interest that senescing CPs secrete Chl-containing globules indicating that some CP components are broken down outside the CP. Senescence appears to be imposed on the CP by the nucleus, and all the known senescence-altering genes except one, cytG in soybean, are nuclear. Only the d₁d₂ mutation(s) in soybean prevents a broad range of leaf senescence processes. Exactly, what causes cell death is unclear; however, the selective thiol protease inhibitor, E-64, does delay death, and this suggests that proteases play a key role.     

A homolog of the defender against apoptotic death gene (DAD1) in senescing gladiolus petals is down-regulated prior to the onset of programmed cell death

We isolated a homolog of the potential anti-apoptotic gene, defender against apoptotic death (DAD1) from gladiolus petals as full-length cDNA (GlDAD1), and investigated the relationship between its expression and the execution processes of programmed cell death (PCD) in senescing petals. RNA gel blotting showed that GlDAD1 expression in petals was drastically reduced, considerably before the first visible senescence symptom (petal wilting). A few days after down-regulation GlDAD1 expression, DNA and nuclear fragmentation were observed, both specific for the execution phase of PCD.     

Endonuclease activities in the coleoptile and the first leaf of developing etiolated wheat seedlings

DNase activity in coleoptiles and the first leaf apices of winter wheat (Triticum aestivum L., cv. Mironovskaya 808) etiolated seedlings was found to increase significantly during seedling growth, peaking on the eighth day of plant development. The maximum of DNase activity was coincident with apoptotic internucleosomal DNA fragmentation in these organs. Wheat endonucleases are capable of hydrolyzing both singleand double-stranded DNA of various origins. The leaf and coleoptiles were found to exhibit nuclease activities that hydrolyzed the lambda phage DNA with N⁶-methyladenine and 5-methylcytosine more actively compared to the hydrolysis of similar unmethylated DNAs. Thus, the endonucleases of wheat seedlings are sensitive to the methylation status of their substrate DNAs. The leaves and coleoptiles exhibited both Ca²⁺/Mg²⁺- and Zn²⁺-dependent nuclease activities that underwent differential changes during development and senescence of seedling organs. EDTA at a concentration of 50 mM fully inhibited the total DNase activity. Electrophoretic heterogeneity was observed for DNase activities operating simultaneously in the coleoptile and the first leaf at different stages of seedling development. Proteins exhibiting DNase activity (16–80 kD mol wt) were revealed in the first leaf and the coleoptile; these proteins were mostly nucleases with the pH optimum around 7.0. Some endonucleases (mol wts of 36, 39, and 28 kD) were present in both organs of the seedling. Some other DNases (mol wts of 16, 56, and about 80 kD) were found in the coleoptile; these DNases hydrolyzed DNA in the nucleus at terminal stages of apoptosis. Different suites of DNase activities were revealed in the nucleus and the cytoplasm, the nuclear DNase activities being more diverse than the cytoplasmic ones. Thus, the cellular (organspecific) and subcellular heterogeneity in composition and activities of DNases has been revealed in wheat plants. These DNases undergo specific changes during seedling development, serving at various stages of programmed cell death in seedling tissues.     

Genetic transformation of somatic cells. VIII. The effect of the DNA methylation inhibitor 5-azacytidine on the stability of thymidine kinase-negative and -positive phenotypes of transformant clone cells

A study was made of the effect of an DNA methylation inhibitor 5-azacytidine (azaC) on the frequency of reversion to a thymidine kinase-positive (TK+) phenotype in 5-bromodeoxy-uridine (BrdU)-resistant subclones obtained from clones of Chinese hamster cells transformed by thymidine kinase gene (tk-gene) of Herpes simplex virus type 1 (HSV1). It is shown that in 8 of 15 BrdU-resistant subclones azaC increases 2-1000-fold the frequency of reversion to TK+ phenotype. Variations in the inducibility of reversions to TK+ phenotype indicate that the DNA methylation associated with TK- phenotype affects but differently tk gene of HSV1. Cultivation of TK+ cells of transformant clones in the presence of azaC may lead to stabilization (or decrease in the rate of the loss) of TK+ phenotype, or may not influence the stability of transformant phenotype. The reaction of TK+ cells of transformant clones depends both on genetically determined rate of the loss of TK+ phenotype, and on the structure of transforming DNA introduced to cells. A conclusion is drawn that the TK- phenotype of transformant clone cells arises due to processes which are not associated with methylation of tk gene of HSV1 in spite of the fact that such a methylation may later stabilize significantly the TK- phenotype.     

Involvement of DNA methylation in the regulation of STS10 gene expression in Vitis amurensis

DNA methylation is known to play an important role in various developmental processes and defense mechanisms in plants and other organisms. However, it is not known whether DNA methylation is implicated in the genetic regulation of plant secondary metabolism, including resveratrol biosynthesis. Resveratrol is a naturally occurring polyphenol that is present in grapes, peanuts, and other plant sources, and it exhibits a wide range of valuable biologically active properties. The transformation of the wild-growing grape Vitis amurensis with the oncogene rolB from Agrobacterium rhizogenes has been demonstrated to considerably increase resveratrol production. To investigate whether DNA methylation regulates resveratrol biosynthesis, we treated both rolB transgenic and empty vector control V. amurensis cell cultures with the DNA demethylation agent 5-azacytosine (azaC). The azaC treatment significantly increased stilbene synthase 10 gene (VaSTS10) expression and resveratrol content in the V. amurensis cell cultures. Using bisulfite sequencing, we examined the methylation status of VaSTS10 in cell cultures under normal conditions and after azaC treatment. Both the promoter and 3′-end of the protein coding region of the VaSTS10 gene were hypermethylated (54–67 %) in the control cell culture. The rolB transgenic cell culture had high levels of resveratrol and lower hypermethylation levels of the VaSTS10 gene (20–47 %). The azaC treatment resulted in reduction in the DNA methylation levels in the promoter and coding regions of the VaSTS10 gene in both cell cultures. These data suggest that the DNA methylation may be involved in the control of resveratrol biosynthesis via the regulation of STS genes expression.     

The nucellus degenerates by a process of programmed cell death during the early stages of wheat grain development

The nucellus, which is the maternal tissue of the wheat grain, degenerates during the early stages of development. We have investigated whether or not this degenerative process may be considered as programmed cell death (PCD). The analysis of DNA of tissues dissected from developing wheat (Triticum aestivum L. cv Chinese Spring) grains at 5-20 days post anthesis (dpa) showed the presence of DNA laddering, which is indicative of internucleosomal fragmentation of nuclear DNA, in maternal tissues but not in the endosperm. The TUNEL assay showed in-situ internucleosomal fragmentation of DNA in nuclei of parenchymal and epidermal cells of the nucellus, as well as in the pericarp, during the early stages of grain development (5 dpa). Furthermore, internucleosomal fragmentation of nuclear DNA was observed in nucellar projection cells in the middle stages of grain development (13-18 dpa), thus showing a process of PCD in these maternal tissues. Electron-transmission microscopy analysis allowed the morphology of PCD to be characterized in this plant tissue. Initially, fragmentation of the cytoplasm was observed, the nuclear envelope appeared dilated and to be forming vacuoles, and the content of heterochromatin increased. A progressive degradation of the cytosolic contents and organelles was observed, and the plasma membrane was disrupted. However, the Golgi apparatus remained intact and apparently functional even in the final stages of cell death.     

Apoptosis and DNA degradation induced by 1-methyl-4-phenylpyridinium in neurons.

Apoptosis is a prominent mechanism of programmed cell death in lymphocytes and in cancer cells not previously found in neurons. We have identified apoptosis and internucleosomal DNA degradation in cultures of cerebellar granule neurons. 1-methyl-4-phenylpyridinium, a selective neurotoxin that destroys the dopaminergic nigrostriatal pathway and results in a parkinsonian syndrome, increases the rate of apoptosis and kills cerebellar granule cells in culture via induction of programmed cell death. Inhibition of gene expression in granule cells with cycloheximide prevents the MPP(+)-induced apoptosis and the DNA fragmentation. Our findings demonstrate a new pathway of neuron death and suggest the possibility that neurodegenerative diseases may result from the inappropriate activation of programmed cell death by apoptosis.