Anna Zuppini

Endoplasmic reticulum stress-induced programmed cell death in soybean cells

In animal cells, the endoplasmic reticulum may participate in programmed cell death by sensing and transducing apoptotic signals. In an attempt to analyze the role of the endoplasmic reticulum in plant programmed cell death we investigated the effect of cyclopiazonic acid, a specific blocker of plant endoplasmic reticulum-type IIA Ca2+-pumps, in soybean cells. Cyclopiazonic acid treatment elicited endoplasmic reticulum stress and a biphasic increase in cytosolic Ca2+ concentration, followed by the induction of a cell death program. Cyclopiazonic acid-induced programmed cell death occurred with accumulation of H2O2, cytochrome c release from mitochondria, caspase 9- and caspase 3-like protease activation, cytoplasmic shrinkage and chromatin condensation. Chelation of cytosolic Ca2+ with 1,2-bis(2-aminophenoxy)ethane-N,N,N′,N′-tetraacetic acid (acetoxymethil ester) failed to inhibit cyclopiazonic acid-induced cell death. Taken together, our results provide evidence for a role of the endoplasmic reticulum and mitochondria in regulating cyclopiazonic acid-induced programmed cell death in soybean cells, probably via a cross-talk between the two organelles.

An Endopolygalacturonase from Sclerotinia sclerotiorum Induces Calcium-Mediated Signaling and Programmed Cell Death in Soybean Cells

A basic endopolygalacturonase (PG) isoform, produced early by Sclerotinia sclerotiorum when infecting soybean seedlings, was used to examine the signaling role of the enzyme in aequorin-expressing soybean cells. A cytosolic Ca2+ elevation was induced, with a rapid increase (phase 1) and a very slow decrease (phase 2) of Ca2+ concentration, indicating the involvement of Ca2+ ions in PG signaling. Within 1 h of PG-cell contact a remarkable level of cell death was recorded, significantly higher than the control cell culture turnover. The observed morphological and biochemical changes were indicative of the activation of programmed cell death; in particular, cytochrome c release in the cytoplasm and activation of both caspase 9-like and caspase 3-like proteases were found. When a polygalacturonase-inhibiting protein (PGIP) and the PG were simultaneously applied to cells, both the Ca2+ increase and cell death were annulled. The possible roles of prolonged sustained cytosolic Ca2+ concentrations in inducing cell death and of the PG-PGIP interaction in preventing PG signaling are discussed.

Calcium-mediated perception and defense responses activated in plant cells by metabolite mixtures secreted by the biocontrol fungus Trichoderma atroviride

BackgroundCalcium is commonly involved as intracellular messenger in the transduction by plants of a wide range of biotic stimuli, including signals from pathogenic and symbiotic fungi. Trichoderma spp. are largely used in the biological control of plant diseases caused by fungal phytopathogens and are able to colonize plant roots. Early molecular events underlying their association with plants are relatively unknown.ResultsHere, we investigated the effects on plant cells of metabolite complexes secreted by Trichoderma atroviride wild type P1 and a deletion mutant of this strain on the level of cytosolic free Ca2+ and activation of defense responses. Trichoderma culture filtrates were obtained by growing the fungus alone or in direct antagonism with its fungal host, the necrotrophic pathogen Botrytis cinerea, and then separated in two fractions (>3 and <3 kDa). When applied to aequorin-expressing soybean (Glycine max L.) cell suspension cultures, Trichoderma and Botrytis metabolite mixtures were distinctively perceived and activated transient intracellular Ca2+ elevations with different kinetics, specific patterns of intracellular accumulation of reactive oxygen species and induction of cell death. Both Ca2+ signature and cellular effects were modified by the culture medium from the knock-out mutant of Trichoderma, defective for the production of the secreted 42 kDa endochitinase.ConclusionNew insights are provided into the mechanism of interaction between Trichoderma and plants, indicating that secreted fungal molecules are sensed by plant cells through intracellular Ca2+ changes. Plant cells are able to discriminate signals originating in the single or two-fungal partner interaction and modulate defense responses.

Programmed Cell Death and Adaptation: Two Different Types of Abiotic Stress Response in a Unicellular Chlorophyte

Eukaryotic microalgae are highly suitable biological indicators of environmental changes because they are exposed to extreme seasonal fluctuations. The biochemical and molecular targets and regulators of key proteins involved in the stress response in microalgae have yet to be elucidated. This study presents morphological and biochemical evidence of programmed cell death (PCD) in a low temperature strain of Chlorella saccharophila induced by exposure to NaCl stress. Morphological characteristics of PCD, including cell shrinkage, detachment of the plasma membrane from the cell wall, nuclear condensation and DNA fragmentation, were observed. Additionally, a significant production of H(2)O(2) and increase in caspase 3-like activity were detected. We demonstrated that singly applied environmental stresses such as warming or salt stress trigger a pathway of PCD. Intriguingly, the prior application of salt stress seems to reduce heat shock-induced cell death significantly, suggesting a combined effect which activates a defense mechanism in algal cells. These results suggest that C. saccharophila can undergo PCD under stress conditions, and that this PCD shares several features with metazoan PCD. Moreover, the simultaneous exposure of this unicellular chlorophyte to different abiotic stresses results in a tolerance mechanism.

Monitoring programmed cell death triggered by mild heat shock in soybean-cultured cells

Programmed cell death (PCD) is a common form of cellular demise during plant response to environmental stresses. The pathway of PCD has been partially clarified in plants although the underlying molecular mechanisms are still poorly defined. We have investigated the signalling cascade induced by a mild heat treatment causing PCD in soybean cells (Glycine max L.). The data show that heat shock led to the onset of PCD in soybean cells involving H2O2 production and mitochondrial damage. Cytochrome c release accompanies the presence of caspase 9-like and caspase 3-like protease activities. Concomitantly, cells were severely damaged with a progressive cell shrinkage, chloroplast alteration and detachment of the plasma membrane from the cell wall. Chromatin condensation and DNA damage were observed. It is proposed that a mild heat stress induces PCD in soybean cells through a caspase-like-dependent pathway.

Chlorella saccharophila cytochrome f and its involvement in the heat shock response

Cytochrome f is an essential component of the major redox complex of the thylakoid membrane. Cloning and characterization are presented here of a novel partial cDNA (ChspetA) encoding cytochrome f in the psychrophile unicellular green alga Chlorella saccharophila and its involvement in the heat shock (HS) response pathway has been analysed. Semi-quantitative reverse transcriptase PCR analysis showed that ChspetA expression is up-regulated in heat-shocked cells and the protein profile of cytochrome f highlighted a release of cytochrome f into the cytosol depending on the time lapse from the HS. Evans Blue assay, analysis of chromatin condensation, and chloroplast alterations showed the induction of cell death in cell suspensions treated with cytosolic extracts from heat-shocked cells. This study identifies cytochrome f in C. saccharophila that seems to be involved in the HS-induced programmed cell death process. The data suggest that cytochrome f fulfils its role through a modulation of its transcription and translation levels, together with its intracellular localization. This work focuses on a possible role of cytochrome f into the programmed cell death-like process in a unicellular chlorophyte and suggests the existence of chloroplast-mediated programmed cell death machinery in an organism belonging to one of the primary lineages of photosynthetic eukaryotes.

Calreticulin and the Endoplasmic Reticulum in Plant Cell Biology

Calreticulin is ubiquitously expressed in plants. The plant homologue shares with its animal counterpart a similar structural organization and basic functioning. A wide range of developmental and environmental stimuli differentially affect the expression of calreticulin in plant cells, highlighting its importance in cell physiology. Nevertheless, current knowledge on calreticulin’s relevance in plant physiology is rather limited compared with animal systems. The contribution of the endoplasmic reticulum to Ca2+ homeostasis and signalling, and the multifunctional role of calreticulin in plant cellular events are rapidly emerging areas of study in plant biology.

CA2+ BINDING PROTEIN CALRETICULIN IN CHLAMYDOMONAS REINHARDTII (CHLOROPHYTA): BIOCHEMICAL CHARACTERIZATION, DIFFERENTIAL EXPRESSION DURING SEXUAL REPRODUCTION, AND PHYLOGENETIC ANALYSIS

The occurrence of calreticulin, the main Ca2+ binding protein in the endoplasmic reticulum of eukaryotic cells, was investigated in the unicellular green alga Chlamydomonas reinhardtii Dangeard. The biochemical characterization of a diethylaminoethyl purified extract highlighted the presence, on SDS‐PAGE, of a 55‐kDa protein that stained blue with the Stains All dye, a diagnostic feature of acidic Ca2+ binding proteins. Immunoblot analyses revealed a strong cross‐reaction of the Chlamydomonas reinhardtii protein with antibodies to plant calreticulins and the endoplasmic reticulum retention signal HDEL. Furthermore, the 55‐kDa protein bound [45Ca2+] and had an acidic isoelectric point (pI = 4.9) but was neither glycosylated nor phosphorylated. N‐terminal sequencing revealed strong amino acid sequence similarity to calreticulin from other sources. The presence of calreticulin in Chlamydomonas reinhardtii suggested that an endoplasmic reticulum Ca2+ buffering mechanism was present in this unicellular chlorophyte. The data suggest an early origin and high conservation of endoplasmic‐reticulum‐mediated Ca2+ functions in eukaryotes, whereby specific posttranslational modifications of the proteinhave been specifically acquired in different lineages of photosynthetic eukaryotes. Moreover, northern and western blot analysis experiments showed a regulation of calreticulin expression during Chlamydomonas sexual reproduction with a high abundance of calreticulin mRNA and protein in reproductive cells.