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Programmed cell death (PCD) occurs in plants during development and defense, but the processes and mechanisms are not yet defined. Culture of carrot single cells at a cell density of

The Fusarium genus of fungi is responsible for commercially devastating crop diseases and the contamination of cereals with harmful mycotoxins. Fusarium mycotoxins aid infection, establishment, and spread of the fungus within the host plant. We investigated the effects of the Fusarium mycotoxin deoxynivalenol (DON) on the viability of Arabidopsis cells. Although it is known to trigger apoptosis in animal cells, DON treatment at low concentrations surprisingly did not kill these cells. On the contrary, we found that DON inhibited apoptosis-like programmed cell death (PCD) in Arabidopsis cells subjected to abiotic stress treatment in a manner independent of mitochondrial cytochrome c release. This suggested that Fusarium may utilise mycotoxins to suppress plant apoptosis-like PCD. To test this, we infected Arabidopsis cells with a wild type and a DON-minus mutant strain of F. graminearum and found that only the DON producing strain could inhibit death induced by heat treatment. These results indicate that mycotoxins may be capable of disarming plant apoptosis-like PCD and thereby suggest a novel way that some fungi can influence plant cell fate. Science Foundation Ireland

In Arabidopsis thaliana we demonstrate that dying root hairs provide an easy and rapid in vivo model for the morphological identification of apoptotic-like programmed cell death (AL-PCD) in plants. The model described here is transferable between species, can be used to investigate rates of AL-PCD in response to various treatments and to identify modulation of AL-PCD rates in mutant/transgenic plant lines facilitating rapid screening of mutant populations in order to identify genes involved in AL-PCD regulation.

Sphingolipids are ubiquitous components of eukaryotic cells and sphingolipid metabolites, such as the long chain base phosphate (LCB-P), sphingosine 1 phosphate (S1P) and ceramide (Cer) are important regulators of apoptosis in animal cells. This study evaluated the role of LCB-Ps in regulating apoptotic-like programmed cell death (AL-PCD) in plant cells using commercially available S1P as a tool. Arabidopsis cell cultures were exposed to a diverse array of cell death-inducing treatments (including Cer) in the presence of S1P. Rates of AL-PCD and cell survival were recorded using vital stains and morphological markers of AL-PCD. Internal LCB-P levels were altered in suspension cultured cells using inhibitors of sphingosine kinase and changes in rates of death in response to heat stress were evaluated. S1P reduced AL-PCD and promoted cell survival in cells subjected to a range of stresses. Treatments with inhibitors of sphingosine kinase lowered the temperature which induced maximal AL-PCD in cell cultures. The data supports the existence of a sphingolipid rheostat involved in controlling cell fate in Arabidopsis cells and that sphingolipid regulation of cell death may be a shared feature of both animal apoptosis and plant AL-PCD.

Programmed cell death (PCD) describes a small number of processes that result in a highly controlled, and organised, form of cellular destruction, activated in every part of the plant, throughout its entire life cycle. For example, PCD is a critical component of many vegetative and reproductive developmental processes, senescence programmes, pathogen defence mechanisms and stress responses. Cell destruction can manifest as apoptotic-like, necrotic or autophagic cell death, and these processes are likely to overlap extensively, sharing several regulatory mechanisms. Several of the key PCD regulators and signals have now been revealed, for example, many cell organelles, including mitochondria, chloroplasts, Golgi apparatus, endoplasmic reticulum and vacuoles have been shown to have a role in controlling PCD activation. Following activation the actual dismantling of the cell appears to involve cell death proteases including those with caspase-like, or metacaspase, activity. This review will examine the current state of knowledge about the regulation of events during plant PCD. We will describe numerous examples of developmental or environmentally induced deaths and outline their potential as model systems for use in PCD research programmes. Similarly, a range of techniques and in vitro model systems that can be used to identify, and quantify, rates of plant PCD are reviewed. These model systems and techniques can be used to identify the underlying signals and events that drive and regulate PCD and ultimately reveal the steps necessary for the botanical dance of death.

Chloroplasts produce reactive oxygen species (ROS) during cellular stress. ROS are known to act as regulators of programmed cell death (PCD) in plant and animal cells, so it is possible that chloroplasts have a role in regulating PCD in green tissue. Arabidopsis thaliana cell suspension cultures are model systems in which to test this, as here it is shown that their cells contain well-developed, functional chloroplasts when grown in the light, but not when grown in the dark. Heat treatment at 55 degrees C induced apoptotic-like (AL)-PCD in the cultures, but light-grown cultures responded with significantly less AL-PCD than dark-grown cultures. Chloroplast-free light-grown cultures were established using norflurazon, spectinomycin, and lincomycin and these cultures responded to heat treatment with increased AL-PCD, demonstrating that chloroplasts affect AL-PCD induction in light-grown cultures. Antioxidant treatment of light-grown cultures also resulted in increased AL-PCD induction, suggesting that chloroplast-produced ROS may be involved in AL-PCD regulation. Cycloheximide treatment of light-grown cultures prolonged cell viability and attenuated AL-PCD induction; however, this effect was less pronounced in dark-grown cultures, and did not occur in antioxidant-treated light-grown cultures. This suggests that a complex interplay between light, chloroplasts, ROS, and nuclear protein synthesis occurs during plant AL-PCD. The results of this study highlight the importance of taking into account the time-point at which cells are observed and whether the cells are light-grown and chloroplast-containing or not, for any study on plant AL-PCD, as it appears that chloroplasts can play a significant role in AL-PCD regulation.

In plants, apoptotic-like programmed cell death (PCD) can be distinguished from other forms of plant cell death by protoplast condensation that results in a morphologically distinct cell corpse. In addition, there is a central regulatory role for the mitochondria and the degradation of the cell and its contents by PCD associated proteases. These distinguishing features are shared with animal apoptosis as it is probable that plant and animal cell death programmes arose in a shared unicellular ancestor. However, animal and plant cell death pathways are not completely conserved. The cell death programmes may have been further modified after the divergence of plant and animal lineages leading to converged, or indeed unique, features of their respective cell death programmes. In this review we will examine the features of apoptotic-like PCD in plants and examine the probable conserved components such as mitochondrial regulation through the release of apoptogenic proteins from the mitochondrial intermembrane space, the possible conserved or converged features such as “caspase-like” molecules which drive cellular destruction and the emerging unique features of plant PCD such as chloroplast involvement in cell death regulation.

Programmed cell death (PCD) is now accepted as a fundamental cellular process in plants. It is involved in defence, development and response to stress, and our understanding of these processes would be greatly improved through a greater knowledge of the regulation of plant PCD. However, there may be several types of PCD that operate in plants, and PCD research findings can be confusing if they are not assigned to a specific type of PCD. The various cell-death mechanisms need therefore to be carefully described and defined. This review describes one of these plant cell death processes, namely the apoptotic-like PCD (AL-PCD). We begin by examining the hallmark 'apoptotic-like' features (protoplast condensation, DNA degradation) of the cell's destruction that are characteristic of AL-PCD, and include examples of AL-PCD during the plant life cycle. The review explores the possible cellular 'executioners' (caspase-like molecules; mitochondria; de novo protein synthesis) that are responsible for the hallmark features of the cellular destruction. Finally, senescence is used as a case study to show that a rigorous definition of cell-death processes in plant cells can help to resolve arguments that occur in the scientific literature regarding the timing and control of plant cell death.

The chloroplast genome encodes a number of proteins, including thylakoid proteins and the large subunit of ribulose biphosphate carboxylase, associated with the structure and function of the chloroplast (1-2). In addition, many components of the chloroplast translational machinery, such as all of the RNAs and some of the ribosomal proteins, are coded by the chloroplast DNA. Although there have been numerous investigations into the genetics of algal chloroplasts, similar studies with higher plants have been hampered by the uniparental (maternal) pattern of transmission of chloroplasts observed in most species, and the shortage of suitable genetic markers (3,4).

Peroxidases and oxalate oxidases are enzymes associated with the production and scavenging of hydrogen peroxide in plants. Peroxidases can influence developmental processes, including auxin metabolism (Normanly, 1997) and lignification (Mader and Amberg-Fisher, 1982), as well as responses to biotic (Thordal-Christensen et al., 1992) and abiotic stresses (Jansen et al., 2001). Oxalate oxidase (germin) is although believed to have several roles in plant development and response to pathogens (Bernier and Berna, 2001), and has recently been shown to afford resistance to insect predation (Ramputh et al., 2002). Hydrogen peroxide (H2O2), levels might reasonably be expected to be altered by modifications in activities of peroxidase (H2O2-utilising) and oxalate oxidase (H2O2-generating) enzymes. H2O2 itself may act as a signaling molecule, triggering programmed cell death and the production of lignified cells. This may be defence related, as in the “oxidative burst” associated with hypersensitive response to pathogens (Levine et al., 1994), or connected to normal developmental processes, such as xylogenesis. The latter involves both programmed cell death, and the action of peroxidase in the cross-linking of lignin precursors.

In plants, cells differentiate according to their position with relation to their cell neighbours. Monoclonal antibody (MAb) probes to polysaccharide epitopes, present at the surfaces of all plant cells, have defined a family of proteoglycan antigens which signify cellular position. These MAbs have been used to sort the single cells present in carrot somatic cell cultures on the basis of the presence or absence of specific polysaccharide epitopes. This sorting allows embryo initial cells to be cultured among different cell collectives (based on their polysaccharide epitope expression) and thus in altered contextual backgrounds. These experiments have shown that specific populations of embryo initial precursor cells induce and sustain the early development of the embryo initials, revealing that the populations of different cell collectives which are defined by different polysaccharide epitopes (cell-context) serves important regulatory function in early plant development. Somatic embryo initials deprived of the influence of the cell collective — defined by the presence of the polysaccharide epitope recognised by the MAb JIM8 — establish unorganised first divisions and develop as callus. However, in the presence of the JIM8-reactive cell collective, or medium conditioned by the collective, the initials develop into somatic embryos. This demonstrates that the cells defined by the JIM8 polysaccharide epitope are necessary to sustain the meristematic activity which drives the renewed development. Transfer of a cell-wall signal from the JIM8- reactive cells to cellular situations in carrot seedlings in which they would not normally occur (out-of-context signals) stimulates lateral root production, thus demonstrating that the inductive signal operative in suspension cultures can be reinterpreted by specific cells later in development and reinitiate meristematic activity. The communication between the precursor cells defined by JIM8 and embryo initials defines an early cell-cell interaction in developing carrot plants. Labelling of flower sections suggests that the same interaction exists between embryo apical and basal cells early in normal development.

A number of Solanum nigrum mutants resistant to the antibiotics spectinomycin, streptomycin and lincomycin have been isolated from regenerating leaf strips after mutagenesis with nitroso-methylurea. Selection of streptomycin- and spectinomycin-resistant mutants has been described earlier. Lincomycin-resistant mutants show resistance to higher levels of the antibiotic than used in the initial selection, and in the most resistant mutant (Ll7A1) maternal inheritance of the trait was demonstrated. The lincomycin-resistant mutant L17A1 and a streptomycin plus spectinomycin resistant double mutant (StSpl) were chosen for detailed molecular characterisation. Regions of the plastid DNA, within the genes encoding 16S and 23S rRNA and rps12 (3′) were sequenced. For spectinomycin and lincomycin resistance, base changes identical to those in similar Nicotiana mutants were identified. Streptomycin resistance is associated with an A → C change at codon 87 of rps 12 (converting a lysine into a glutamine), three codons upstream from a mutation earlier reported for Nicotiana. This site has not previously been implicated in streptomycin resistance mutations of higher plants, but has been found in Escherichia coli. The value of these mutants for studies on plastid genetics is discussed.

Cells in a plant differentiate according to their positions and use cell-cell communication to assess these positions. Similarly, single cells in suspension cultures can develop into somatic embryos, and cell-cell communication is thought to control this process. The monoclonal antibody JIM8 labels an epitope on cells in specific positions in plants. JIM8 also labels certain cells in carrot embryogenic suspension cultures. We have used JIM8 and secondary antibodies coupled to paramagnetic beads to label and immunomagnetically sort single cells in a carrot embryogenic suspension culture into pure populations. Cells in the JIM8(+) population develop into somatic embryos, whereas cells in the JIM8(-) population do not form somatic embryos. However, certain cells in JIM8(+) cultures (state B cells) undergo asymmetric divisions, resulting in daughter cells (state C cells) that do not label with JIM8 and that sort to JIM8(-) cultures. State C cells are competent to form somatic embryos, and we show here that a conditioned growth medium from a culture of JIM8(+) cells allows state C cells in a JIM8(-) culture to go on and develop into somatic embryos. JIM8 labels cells in suspension cultures at the cell wall. Therefore, a cell with a role in cell-cell communication and early cell fate selection can be identified by an epitope in its cell wall.

Summary Monoclonal antibodies which recognize carbohydrate in arabinogalactan proteins (AGPs) have revealed that certain carbohydrate epitopes at the outer plasma membrane surface are demonstratively regulated. Some epitomes are expressed according to cell position, and AGES are thought to play a role in cell—cell interaction during development. This study demonstrates that sugar beet plasma membranes contain two subagencies of AGES, with apparent molecular masses of 82 and 97 kDa, and that each subfamily consists of a small number of acidic AGP isoforms. Excision of leaves generates three additional AGP complexes with apparent molecular masses of 120, 170 and 210 kDa, with the 170 kDa complex being the major form induced by excision. The addition of millimolar concentrations of H2O2 to a partially purified fraction of the 82 and 97 kDa AGPs also generates AGP complexes, with the 170 kDa complex as the major form. These results indicate that the plasma membrane AGPs are a target for endogenous H2O2.