Ernst J. Woltering

Morphological classification of plant cell deaths

Programmed cell death (PCD) is an integral part of plant development and of responses to abiotic stress or pathogens. Although the morphology of plant PCD is, in some cases, well characterised and molecular mechanisms controlling plant PCD are beginning to emerge, there is still confusion about the classification of PCD in plants. Here we suggest a classification based on morphological criteria. According to this classification, the use of the term ‘apoptosis’ is not justified in plants, but at least two classes of PCD can be distinguished: vacuolar cell death and necrosis. During vacuolar cell death, the cell contents are removed by a combination of autophagy-like process and release of hydrolases from collapsed lytic vacuoles. Necrosis is characterised by early rupture of the plasma membrane, shrinkage of the protoplast and absence of vacuolar cell death features. Vacuolar cell death is common during tissue and organ formation and elimination, whereas necrosis is typically found under abiotic stress. Some examples of plant PCD cannot be ascribed to either major class and are therefore classified as separate modalities. These are PCD associated with the hypersensitive response to biotrophic pathogens, which can express features of both necrosis and vacuolar cell death, PCD in starchy cereal endosperm and during self-incompatibility. The present classification is not static, but will be subject to further revision, especially when specific biochemical pathways are better defined.

Physiology and molecular biology of petal senescence

Petal senescence is reviewed, with the main emphasis on gene expression in relation to physiological functions. Autophagy seems to be the major mechanism for large-scale degradation of macromolecules, but it is still unclear if it contributes to cell death. Depending on the species, petal senescence is controlled by ethylene or is independent of this hormone. EIN3-like (EIL) transcription factors are crucial in ethylene-regulated senescence. The presence of adequate sugar levels in the cell delays senescence and prevents an increase in the levels of EIL mRNA and the subsequent up-regulation of numerous senescence-associated genes. A range of other transcription factors and regulators are differentially expressed in ethylene-sensitive and ethylene-insensitive petal senescence. Ethylene-independent senescence is often delayed by cytokinins, but it is still unknown whether these are natural regulators. A role for caspase-like enzymes or metacaspases has as yet not been established in petal senescence, and a role for proteins released by organelles such as the mitochondrion has not been shown. The synthesis of sugars, amino acids, and fatty acids, and the degradation of nucleic acids, proteins, lipids, fatty acids, and cell wall components are discussed. It is claimed that there is not enough experimental support for the widely held view that a gradual increase in cell leakiness, resulting from gradual plasma membrane degradation, is an important event in petal senescence. Rather, rupture of the vacuolar membrane and subsequent rapid, complete degradation of the plasma membrane seems to occur. This review recommends that more detailed analysis be carried out at the level of cells and organelles rather than at that of whole petals.

Do plant Caspases exist

Programmed cell death (PCD) is a functional concept that refers to cell death that is part of the normal life of a multicellular organism; it involves controlled disassembly of the cell. In animal systems PCD is synonymous with apoptosis, a cell death process characterized by a distinct set of

Ethylene biosynthetic genes are differentially expressed during carnation (Dianthus caryophyllus L.) flower senescence.

Ethylene production and expression patterns of an 1-aminocyclopropane-1-carboxylic acid (ACC) oxidase (CARAO1) and of two ACC synthase (EC 4.4.1.14) genes (CARACC3 and CARAS1) were studied in floral organs of cut carnation flowers (Dianthus caryophyllus L.) cv. White Sim. During the vase life and after treatment of fresh flowers with ethylene, production of ethylene and expression of ethylene biosynthetic genes first started in the ovary followed by the styles and the petals. ACC oxidase was expressed in all the floral organs whereas, during the vase life, tissue-specific expression of the two ACC synthase genes was observed. After treatment with a high ethylene concentration, tissue specificity of the two ACC synthase genes was lost and only a temporal difference in expression remained. In styles, poor correlation between ethylene production and ACC synthase (CARAS1) gene expression was observed suggesting that either activity is regulated at the translational level or that the CARAS1 gene product requires an additional factor for activity.Isolated petals showed no increase in ethylene production and expression of ethylene biosynthetic genes when excised from the flower before the increase in petal ethylene production (before day 7); showed rapid cessation of ethylene production and gene expression when excised during the early phase of petal ethylene production (day 7) and showed a pattern of ethylene production and gene expression similar to the pattern observed in the attached petals when isolated at day 8. The interorgan regulation of gene expression and ethylene as a signal molecule in flower senescence are discussed.

Histochemical and genetic analysis of host and non-host interactions of Arabidopsis with three Botrytis species: an important role for cell death control

SUMMARY Susceptibility was evaluated of host and non-host plants to three pathogenic Botrytis species: the generalist B. cinerea and the specialists B. elliptica (lily) and B. tulipae (tulip). B. tulipae was, unexpectedly, able to infect plant species other than tulip, and to a similar extent as B. cinerea. To study host and non-host interactions in more detail, the three Botrytis species were inoculated on Arabidopsis wild-types and 23 mutant genotypes. Disease development was monitored macroscopically by quantifying the lesion area and microscopically by bright-field and fluorescence microscopy following histochemical staining. B. cinerea and B. tulipae were very similar in their ability to infect the tested Arabidopsis genotypes, whereas B. elliptica caused disease only on a few Arabidopsis mutant genotypes. Arabidopsis mutants with a delayed or reduced cell death response were generally more resistant to Botrytis infection, whereas mutants in which cell death was accelerated were more susceptible. Differences in susceptibility between genotypes were generally gradual. Only the camalexin-deficient mutant pad3 was fully susceptible to all three Botrytis species. Cellular changes were monitored during compatible and incompatible interactions. The formation of papillae, the presence of lysosome-like vesicles and the intracellular accumulation of H(2)O(2) and nitric oxide were visualized in the infection zones using fluorescent probes. Based on histology and responses of Arabidopsis mutants, a model is proposed in which resistance against Botrytis, besides the production of camalexin, depends on the balance between cell death and survival.

Ethylene perception is required for the expression of tomato ripening-related genes and associated physiological changes even at advanced stages of ripening

Treatment of tomato fruit (Lycopersicon esculentum L. cv Prisca) with 1-methylcyclopropene (1-MCP), a potent inhibitor of ethylene action, delayed colour development, softening, and ethylene production in tomato fruit harvested at the mature green breaker, and orange stages. 1-MCP treatment also decreased the mRNA abundance of phytoene synthase 1 (PSY1), expansin 1 (EXP1), and 1-aminocyclopropane-1-carboxylic acid (ACC) oxidase 1 (ACO1), three ripening-related tomato genes, in mature green, breaker, orange, and red ripe fruit. These results demonstrate that the ripening process can be inhibited both on a physiological and molecular level, even at very advanced stages of ripening. The effects of 1-MCP on ripening lasted 5–7 days and could be prolonged by renewed exposure.

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

Abstract. 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.

A tomato metacaspase gene is upregulated during programmed cell death in Botrytis cinerea -infected leaves

Programmed cell death (PCD) in plant cells is often accompanied by biochemical and morphological hallmarks similar to those of animal apoptosis. However, orthologs of animal caspases, cysteinyl aspartate-specific proteases that constitute the core component of animal apoptosis, have not yet been identified in plants. Recent studies have revealed the presence of a family of genes encoding proteins with distant homology to mammalian caspases, designated metacaspases, in the Arabidopsis thaliana genome. Here, we describe the isolation of LeMCA1, a type-II metacaspase cDNA clone from tomato (Lycopersicon esculentum Mill.). BLAST analysis demonstrated that the LeMCA1 gene is located in close vicinity of several genes that have been linked with PCD. Southern analysis indicated the existence of at least one more metacaspase in the tomato genome. LeMCA1 mRNA levels rapidly increased upon infection of tomato leaves with Botrytis cinerea, a fungal pathogen that induces cell death in several plant species. LeMCA1 was not upregulated during chemical-induced PCD in suspension-cultured tomato cells.

Molecular cloning of two different ACC synthase PCR fragments in carnation flowers and organ specific expression of the corresponding genes.

Degenerate oligonucleotides to highly conserved regions of 1-aminocyclopropane-1-carboxylic acid (ACC) synthase (EC 4.4.1.14), the key enzyme in ethylene biosynthesis, were used to prime the synthesis and amplification of fragments of about 1180 bp by polymerase chain reaction (PCR) in samples of cDNA to total RNA isolated from senescing carnation (Dianthus caryophyllus) flowers. Two putative ACC synthase PCR clones were isolated one of which was identical to the sequence of a carnation ACC synthase cDNA clone (CARACC3) recently isolated by Park et al. (Plant Mol Biol 18 (1992) 377–386). The other clone (CARAS1) was ca. 66% homologous at the amino acid level to CARACC3. For both ACC synthase clones, specific oligonucleotides were synthesized and, using PCR, we were able to distinguish between the two ACC synthase transcripts in samples of total RNA isolated from different carnation flower parts and leaves.DNA blots of PCR fragments revealed that, in flowers, both ageing and ethylene stimulated the occurrence of these transcripts in an organ-specific way. CARACC3 was more abundant in RNA from the petals whereas CARAS1 was more abundant in RNA from the styles. Despite a high ethylene production observed in ovaries, the level of both transcripts was low, suggesting the existence of a third ACC synthase gene that is specifically expressed in the ovary. Transcript levels in leaves were low irrespective of treatment.

A tomato homologue of the human protein PIRIN is induced during programmed cell death

Programmed cell death (PCD), with similarities to animal apoptosis, was induced in tomato suspension cells by the topoisomerase I inhibitor camptothecin. Previously, a differential display screening was performed to isolate genes differentially expressed during camptothecin-induced cell death. As a result, the new tomato gene Le-pirin was isolated, whose mRNA levels dramatically increase during camptothecin-induced PCD. Le-pirin mRNA accumulation is also observed when cell death is triggered by the mycotoxin fumonisin-B1, but not when the suspension cells are treated with stress-related compounds such as ethylene, methyl jasmonate or salicylic acid. The caspase inhibitor Z-Asp-CH2-DCB and the calcium channel blocker LaCl3 effectively delayed whereas ethylene greatly stimulated camptothecin-induced PCD and the accumulation of Le-pirin mRNA. The Le-pirin encoded protein shows 56% identity with the human protein PIRIN, a nuclear factor reported to interact with the human oncogene Bcl-3. Human PIRIN stabilizes the formation of quaternary complexes between Bcl-3, the anti-apoptotic transcription factor NF-κB and its DNA target sequences in vitro. The isolation of Le-pirin and its implication in plant PCD provides new clues on the role of putative NF-κB-associated pathways in plant defence mechanisms.