Barbara Baldan

Phosphate deprivation induces transfer of DGDG galactolipid from chloroplast to mitochondria

In many soils plants have to grow in a shortage of phosphate, leading to development of phosphate-saving mechanisms. At the cellular level, these mechanisms include conversion of phospholipids into glycolipids, mainly digalactosyldiacylglycerol (DGDG). The lipid changes are not restricted to plastid membranes where DGDG is synthesized and resides under normal conditions. In plant cells deprived of phosphate, mitochondria contain a high concentration of DGDG, whereas mitochondria have no glycolipids in control cells. Mitochondria do not synthesize this pool of DGDG, which structure is shown to be characteristic of a DGD type enzyme present in plastid envelope. The transfer of DGDG between plastid and mitochondria is investigated and detected between mitochondria-closely associated envelope vesicles and mitochondria. This transfer does not apparently involve the endomembrane system and would rather be dependent upon contacts between plastids and mitochondria. Contacts sites are favored at early stages of phosphate deprivation when DGDG cell content is just starting to respond to phosphate deprivation.

A Diffusible Signal from Arbuscular Mycorrhizal Fungi Elicits a Transient Cytosolic Calcium Elevation in Host Plant Cells

The implication of calcium as intracellular messenger in the arbuscular mycorrhizal (AM) symbiosis has not yet been directly demonstrated, although often envisaged. We used soybean (Glycine max) cell cultures stably expressing the bioluminescent Ca2+ indicator aequorin to detect intracellular Ca2+ changes in response to the culture medium of spores of Gigaspora margarita germinating in the absence of the plant partner. Rapid and transient elevations in cytosolic free Ca2+ were recorded, indicating that diffusible molecules released by the mycorrhizal fungus are perceived by host plant cells through a Ca2+-mediated signaling. Similar responses were also triggered by two Glomus isolates. The fungal molecules active in generating the Ca2+ transient were constitutively released in the medium, and the induced Ca2+ signature was not modified by the coculture of germinating spores with plant cells. Even ungerminated spores were able to generate the signaling molecules, as proven when the germination was blocked by a low temperature. The fungal molecules were found to be stable to heat treatment, of small molecular mass (<3 kD), and, on the basis of extraction with an organic solvent, partially lipophilic. Evidence for the specificity of such an early fungal signal to the AM symbiosis is suggested by the lack of a Ca2+ response in cultured cells of the nonhost plant Arabidopsis (Arabidopsis thaliana) and by the up-regulation in soybean cells of genes related to Medicago truncatula DMI1, DMI2, and DMI3 and considered essential for the establishment of the AM symbiosis.

Coexistence of predominantly nonculturable rhizobia with diverse, endophytic bacterial taxa within nodules of wild legumes

A previous analysis showed that Gammaproteobacteria could be the sole recoverable bacteria from surface-sterilized nodules of three wild species of Hedysarum. In this study we extended the analysis to eight Mediterranean native, uninoculated legumes never previously investigated regarding their root-nodule microsymbionts. The structural organization of the nodules was studied by light and electron microscopy, and their bacterial occupants were assessed by combined cultural and molecular approaches. On examination of 100 field-collected nodules, culturable isolates of rhizobia were hardly ever found, whereas over 24 other bacterial taxa were isolated from nodules. None of these nonrhizobial isolates could nodulate the original host when reinoculated in gnotobiotic culture. Despite the inability to culture rhizobial endosymbionts from within the nodules using standard culture media, a direct 16S rRNA gene PCR analysis revealed that most of these nodules contained rhizobia as the predominant population. The presence of nodular endophytes colocalized with rhizobia was verified by immunofluorescence microscopy of nodule sections using an Enterobacter-specific antibody. Hypotheses to explain the nonculturability of rhizobia are presented, and pertinent literature on legume endophytes is 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.

Primary structure of the N-linked carbohydrate chains of Calreticulin from spinach leaves.

Calreticulin is a multifunctional Ca2+-binding protein of the endoplasmic reticulum of most eukaryotic cells. The 56 kDa Calreticulin glycoprotein isolated from spinach (Spinacia oleracea L.) leaves was N-deglycosylated by PNGase-F digestion. The carbohydrate moiety was isolated by gel permeation chromatography and purified by high-pH anion-exchange chromatography. The fractions were investigated by 500 MHz1H-NMR spectroscopy, in combination with monosaccharide analysis and fast-atom bombardment-mass spectrometry. The following carbohydrate structure could be established as the major component (Man8GlcNAc2): Heterogeneity was demonstrated by the presence of two minor components being Man7GlcNAc2 lacking a terminal residue (D1 or D3), compared to the major component. A cross-reactivity with an antibody against the endoplasmic reticulum retention signal HDEL was also found.

Phosphate availability affects the tonoplast localization of PLDζ2, an Arabidopsis thaliana phospholipase D

Under phosphate deprivation, higher plants change their lipid composition and recycle phosphate from phospholipids. A phospholipase D, PLDζ2, is involved in this recycling and in other cellular functions related to plant development. We investigated the localization of Arabidopsis PLDζ2 by cell fractionation and in vivo GFP confocal imaging. AtPLDζ2 localizes to the tonoplast and the Nter regulatory domain is sufficient for its sorting. Under phosphate deprivation, AtPLDζ2 remains located in the tonoplast but its distribution is uneven. We observed PLDζ2‐enriched tonoplast domains preferentially positioned close to mitochondria and beside chloroplasts. In absence of PLDζ2, membrane developments were visualized inside vacuoles.

Synthesis of chlorophyll and photosynthetic competence in etiolated and greening seedlings of Larix decidua as compared with Picea abies

Summary The development of the photosynthetic apparatus was studied in seedlings of Picea abies and Larix decidua grown both in darkness and in light. In light the pattern of differentiation was similar in the two species, and mature chloroplasts showed a well developed thylakoid system arranged into granal and intergranal regions. In the dark, etio-chloroplasts were formed that had different lamellar and tubuliform membrane amounts in the two species. Spruce chloroplasts contained small prolamellar bodies and many thylakoids; in L. decidua chloroplasts, voluminous prolamellar bodies and only a few thylakoids were formed. Fluorescence emission spectra of cotyledons at 77 K as well as pigment analyses showed different behaviour of L. decidua compared with P. abies when grown in the dark. When etiolated seedlings were exposed to the light, the rate of phototransformation of the etio-chloroplast and the achievement of photosynthetic functionality was fast in spruce and slow in larch. Spruce cotyledons attained normal values of in vivo chlorophyll fluorescence characteristics, i.e. Fv/Fm and quenching coefficients qP and qNP within 1.5 hours of light exposure; with larch, the control value of qP was reached much more quickly (ca 3 h) than that of Fv/Fm (ca 20 h), indicating that PS I becomes fully active before PS II. After exposure to light, etiolated larch seedlings behave like those gymnosperms lacking the ability to synthesize chlorophyll in the dark.

Spontaneous and induced apoptosis in embryogenic cell cultures of carrot (Daucus carota L.) in different physiological states.

Programmed cell death (apoptosis) in plants - and plant cells in culture - has received much less attention than its animal counterpart. In the present work, using agents producing biotic or abiotic stress on cultivated cells from carrot - and, in a few experiments, Arabidopsis -, we show that DNA fragmentation, random or oligonucleosomal, can be induced by different treatments. Moreover, we demonstrate that the same cultures may or may not respond to the inducing signal according to their physiological state. In particular, stationary cells are more responsive to the inducing signal than actively proliferating ones, and cells growing in an unorganized way are more responsive than cells carrying out the embryogenic programme. Senescent cells in culture also appear to die by apoptosis, but healthy cells can also be induced to die apoptotically if exposed to the medium conditioned by senescent cells of the same or different species.

Evidence that Spinach Leaves Express Calreticulin but Not Calsequestrin

The presence of either calreticulin (CR) or calsequestrin (CS)-like proteins in spinach (Spinacia oleracea L.) leaves has been previously described. Here we report the purification from spinach leaves of two highly acidic (isoelectric point 5.2) Ca2+-binding proteins of 56 and 54 kD by means of DEAE-cellulose chromatography followed by phenyl-Sepharose chromatography in the presence of Zn2+ (i.e. under experimental conditions that allowed the purification of CR from human liver). On the other hand, we failed to identify any protein sharing with animal CS the ability to bind to phenyl-Sepharose in the absence of Ca2+. Based on the N-terminal amino acid sequence, the 56- and 54-kD spinach Ca2+-binding proteins were identified as two distinct isoforms of CR. Therefore, we conclude that CR, and not CS, is expressed in spinach leaves. The 56-kD spinach CR isoform was found to be glycosylated, as judged by ligand blot techniques with concanavalin A and affinity chromatography with concanavalin A-Sepharose. Furthermore, the 56-kD CR was found to differ from rabbit liver CR in amino acid sequence, peptide mapping after partial digestion with Staphylococcus aureus V8 protease, pH-dependent shift of electrophoretic mobility, and immunological cross-reactivity with an antiserum raised to spinach CR, indicating a low degree of structural homology with animal CRs.