Programmed cell death during the transition from multicellular structures to globular embryos in barley androgenesis

Androgenesis represents one of the most fascinating examples of cell differentiation in plants. In barley, the conversion of stressed uninucleate microspores into embryo-like structures is highly efficient. One of the bottlenecks in this process is the successful release of embryo-like structures out of the exine wall of microspores. In the present work, morphological and biochemical studies were performed during the transition from multicellular structures to globular embryos. Exine wall rupture and subsequent globular embryo formation were observed only in microspores that divided asymmetrically. Independent divisions of the generative and the vegetative nuclei gave rise to heterogeneous multicellular structures, which were composed of two different cellular domains: small cells with condensed chromatin structure and large cells with normal chromatin structure. During exine wall rupture, the small cells died and their death marked the site of exine wall rupture. Cell death in the small cell domain showed typical features of plant programmed cell death. Chromatin condensation and DNA degradation preceded cell detachment and cytoplasm dismantling, a process that was characterized by the formation of vesicles and vacuoles that contained cytoplasmic material. This morphotype of programmed cell death was accompanied by an increase in the activity of caspase-3-like proteases. The orchestration of such a death program culminated in the elimination of the small generative domain, and further embryogenesis was carried out by the large vegetative domain. To date, this is the first report to show evidence that programmed cell death takes part in the development of microspore-derived embryos.     

Galig, a novel cell death gene that encodes a mitochondrial protein promoting cytochrome c release

Galectin-3 internal gene (Galig) was recently identified as an internal gene transcribed from the second intron of the human galectin-3 gene that is implicated in cell growth, cell differentiation, and cancer development. In this study, we show that galig expression causes morphological alterations in human cells, such as cell shrinkage, cytoplasm vacuolization, nuclei condensation, and ultimately cell death. These alterations were associated with extramitochondrial release of cytochrome c, a known cell death effector. Furthermore, Bcl-xL co-transfection significantly reduced the release of cytochrome c induced by galig expression, suggesting a common pathway between the cytotoxic activity of galig and the anti-apoptotic activity of Bcl-xL. This antagonism was not observed upon co-transfection of Bcl-2 and galig. Galig encodes a mitochondrial-targeted protein named mitogaligin. Structure-activity relationship studies showed that the mitochondrial addressing of mitogaligin relies on an internal sequence that is required and sufficient for the release of cytochrome c and cell death upon cell transfection. Moreover, incubation of isolated mitochondria with peptides derived from mitogaligin induces cytochrome c release. Altogether, these results show that galig is a novel cell death gene encoding mitogaligin, a protein promoting cytochrome c release upon direct interaction with the mitochondria.     

Three dimensional confocal and electron microscopy imaging define the dynamics and mechanisms of diploidisation at early stages of barley microspore-derived embryogenesis

In order to determine the timing and mechanisms of the spontaneous diploidisation throughout microspore-derived embryogenesis in barley, we have estimated the ploidy level of individual nuclei within young pro-embryos, from the first androgenetic division up to multinuclear structures still surounded by the exine. Our methodological approach was based on the measure of the intensity of fluorescence after 4,6-Diamidino-2-phenylindole dihydrochloride staining, nuclear size and number of nucleoli in the confocal microscope. This method avoids the overlapping of the fluorescence signal in multinuclear pro-embryos, which cannot be studied using cytophotometer methods based on other types of fluorescence microscopes. The identification of haploid and diploid nuclei enabled us to determine the timing of diploidisation at early stages throughout androgenetic development. We found that diploidisation is an ongoing process that can start after the first embyogenic division and continues in multinuclear pro-embryos. Reconstruction of 3D-images of entire pro-embryos and the observation of cross and longitudinal sections across stacks of optical sections, together with correlative light and electron microscopy, provided evidences of nuclear fusion as the main mechanism of diploidisation.Electronic Supplementary Material Supplementary material is available for this article at This paper is dedicated to María Ángeles Ollacarizqueta (CCD and Confocal Service of the CIB) on her retirement     

Accumulation of genes for susceptibility to rust fungi for which barley is nearly a nonhost results in two barley lines with extreme multiple susceptibility

Nonhost resistance is the most common type of resistance in plants. Understanding the factors that make plants susceptible or resistant may help to achieve durably effective resistance in crop plants. Screening of 109 barley (Hordeum vulgare L.) accessions in the seedling stage indicated that barley is a complete nonhost to most of the heterologous rust fungi studied, while it showed an intermediate status with respect to Puccinia triticina, P. hordei-murini, P. hordei-secalini, P. graminis f. sp. lolii and P. coronata ff. spp. avenae and holci. Accessions that were susceptible to a heterologous rust in the seedling stage were much more or completely resistant at adult plant stage. Differential interaction between barley accessions and heterologous rust fungi was found, suggesting the existence of rust-species-specific resistance. In particular, many landrace accessions from Ethiopia and Asia, and naked-seeded accessions, tended to be susceptible to several heterologous rusts, suggesting that some resistance genes in barley are effective against more than one heterologous rust fungal species. Some barley accessions had race-specific resistance against P. hordei-murini. We accumulated genes for susceptibility to P. triticina and P. hordei-murini in two genotypes called SusPtrit and SusPmur, respectively. In the seedling stage, these accessions were as susceptible as the host species to the target rusts. They also showed unusual susceptibility to other heterologous rusts. These two lines are a valuable asset to further experimental work on the genetics of resistance to heterologous rust fungi.Electronic Supplementary Material Supplementary material is available in the online version of this article at http://dx.doi.org/10.1007/s00425-004-1319-1Abbreviations ff. spp Formae speciales - RIL Recombinant inbred line - DC Double cross - DC-S Progeny produced by selfing of double-cross plants     

EGF signaling overcomes a uterine cell death associated with temporal mis-coordination of organogenesis within the C. elegans egg-laying apparatus

We isolated cog-3(ku212) as a C. elegans egg-laying defective mutant that is associated with a connection-of-gonad defective phenotype. cog-3(ku212) mutants appear to have no connection between the vulval and the uterine lumens at the appropriate stage because the uterine lumen develops with a temporal delay relative to the vulva and, thus, is not present when the connection normally forms. The lack of temporal synchronization between the vulva and the uterus is not due to precocious or accelerated vulval development. Instead, global gonadogenesis is mildly delayed relative to development of extra-gonadal tissue. cog-3(ku212) mutants also have a specific uterine fate defect. Normally, four cells of the uterine pi lineage respond via their LET-23 epidermal growth factor-like receptors to a vulval-derived LIN-3 EGF signal and adopt the uterine vulval 1 (uv1) fate. In cog-3(ku212) mutants, these four pi progeny cells are set aside as a pre-uv1 population but undergo necrosis prior to full differentiation. A gain-of-function mutation in LET-23 EGF receptor and ectopic expression of LIN-3 EGF within the proper temporal constraints can rescue the uv1 defect, suggesting that a signaling defect, perhaps due to the temporal delay, is at fault. In support of this model, we demonstrate that lack of vulval-uterine coordination due to precocious vulval development also leads to uv1 cell differentiation defects.     

Sphingolipid long chain base phosphates can regulate apoptotic-like programmed cell death in plants

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.     

Role of Cysteine in Enhancing Androgenesis and Regeneration of Indica Rice (Oryza sativa L.)

The effects of amino acid cysteine to culture systems of microspore-derived callus induction as well as plantlet regeneration were studied. Isolated pollen along with anther walls of basmati cultivars, Pusa basmati 1, Basmati 370 and Basmati 386 were cultured in a medium based on N₆ salts supplemented with or without cysteine following pollen embedment in agarose. The induction and regeneration medium with cysteine gave twice as effective androgenesis and plantlet regeneration in recalcitrant basmati rice cultivars as compared with medium lacking cysteine. Unlike the highly responsive model systems, most of the indica cultivars responded rather poorly in anther culture. So the study may accelerate the introgression of desirable genes into basmati rice using anther culture as a breeding tool. Response of microspores in androgenesis, plant regeneration and albinism was genotype specific. Regeneration of Indica rice varieties remains a limiting factor for researchers undertaking transformation experiments.     

Cell cycle dependent distribution of phosphorylated proteins in microspores and pollen ofBrassica napus L., detected by the monoclonal antibody MPM-2

Summary The monoclonal antibody MPM-2, which interacts with a mitosis-specific phosphorylated epitope, has been used to study phosphorylation of proteins in microspores and pollen ofBrassica napus. One- (1-D) and two-dimensional (2-D) immunoblots revealed that MPM-2 recognized a family of phosphorylated proteins in freshly isolated microspores and pollen. The same set of phosphorylated proteins was found after 8 h of culture at embryogenie (32 °C) and non-embryogenic (18 °C) conditions. Two major spots were observed on 2-D immunoblots, one of which (M_r75 kDa, pI5.1) co-localized with the 70 kDa heat shock protein. Immunolabelling of sectioned microspores and pollen showed that MPM-2 reactive epitopes were predominantly observed in the nucleoplasm from G₁ until G2-phase, and in the cytoplasm during mitosis. This may be due to a cell cycle related translocation of phosphoproteins from the nucleus to the cytoplasm, or alternate phosphorylation and dephosphorylation in nucleus and cytoplasm. Detectability of epitopes on sections depended on the embedding procedure. Cryo processing revealed epitope reactivity in all stages of the cell cycle whereas polyethylene glycol embedded material showed no labelling in the cytoplasm during mitosis. Processing might reduce the antigenicity of cytoplasmic MPM-2 detectable proteins, probably due to dephosphorylation. The MPM-2 detectable epitope was observed in all cells investigated, irrespective of culture conditions, and its intracellular distribution depended on the cell cycle stage and was not related to the developmental fate of the microspores and pollen.     

Nucleus substitution between Petunia species using gamma ray-induced androgenesis

Summary The ovaries of two different Petunia species: Petunia hybrida (hort) and Petunia parodii (Steere) were irradiated with -ray doses ranging from 50 to 1,000 Gy before pollination. Seed setting occurred after 4 days preculture on a non-sterile medium. Ovaries transformed into fruits were then cultivated aseptically with the following results: (1) -ray doses ranging from 200 to 1,000 Gy led to the development of two types of plants: haploids 2n=x=7 and overdiploids 2n>2x=14. (2) The androgenetic origin of haploids was ascertained by using genetic markers. The origin of overdiploids is discussed. (3) Androgenetic haploids contained the chloroplasts of the irradiated female parent. No visible change of cp DNA patterns was observed after irradiation. (4) The four possible androgenetic events were successfully obtained between the two Petunia species: hybrida haploids with hybrida or parodii cytoplasm, and parodii haploids with parodii or hybrida cytoplasm.     

The cell wall-plasmalemma interface in sieve tubes of barley

Both thick- and thin-walled sieve tubes in leaf-blade veins of Hordeum vulgare L. exhibit a distinct, electron-opaque inner wall layer after fixation in glutaraldehyde-osmium tetroxide and staining with uranyl acetate and lead citrate. This inner wall layer is thickest at the sieve plates and lateral sieve areas where it is permeated by a labyrinth of tubules formed by the plasmalemma. Along the lateral walls between sieve areas the inner wall layer apparently is penetrated by numerous microvilli-like evaginations of the plasmalemma, giving the cell wall-plasmalemma interface the appearance of a brush border. It is suggested that a similar brush-border-like structure may occur at the cell wall-plasmalemma interface of sieve elements in a wide variety of vascular plants.Abbreviation ER endoplasmic reticulum     

Depletion of the novel protein PHACTR-1 from human endothelial cells abolishes tube formation and induces cell death receptor apoptosis

Using suppression subtractive hybridisation (SSH), we identified a hitherto unreported gene PHACTR-1 (Phosphatase Actin Regulating Protein-1) in Human Umbilical Vascular Endothelial Cells (HUVECs). PHACTR-1 is an actin and protein phosphatase 1 (PP1) binding protein which is reported to be highly expressed in brain and which controls PP1 activity and F-actin remodelling. We have also reported that its expression is dependent of Vascular Endothelial Growth Factor (VEGF-A₁₆₅). To study its function in endothelial cells, we used a siRNA strategy against PHACTR-1. PHACTR-1 siRNA-treated HUVECs showed a major impairment of tube formation and stabilisation. PHACTR-1 depletion triggered apoptosis through death receptors DR4, DR5 and FAS, which was reversed using death receptor siRNAs or with death receptor-dependent caspase-8 siRNA. Our findings suggest that PHACTR-1 is likely to be a key regulator of endothelial cell function properties. Because of its central role in the control of tube formation and endothelial cell survival, PHACTR-1 may represent a new target for the development of anti-angiogenic therapy.     

Rapid and tissue-specific accumulation of solutes in the growth zone of barley leaves in response to salinity

The aim of the present study was to test whether rapid accumulation of solutes in response to salinity in leaf tissues of barley (Hordeum vulgare L.) contributes to recovery and maintenance of residual elongation growth. Addition of 100 mM NaCl to the root medium caused an immediate reduction close to zero in elongation velocity of the growing leaf 3. After 20–30 min, elongation velocity recovered suddenly, to 40–50% of the pre-stress level. Bulk osmolality increased first, after 60 min, significantly in the proximal half of the elongation zone. Over the following 3 days, osmolality increases became significant in the distal half of the elongation zone, the adjacent, enclosed non-elongation zone and finally in the emerged portion of the blade. The developmental gradient and time course in osmolality increase along the growing leaf was reflected in the pattern of solute (Cl, Na and K) accumulation in bulk tissue and epidermal cells. The partitioning of newly accumulated solutes between epidermis and bulk tissue changed with time. Even though solute accumulation does not contribute to the sudden and partial growth recovery 20–30 min after exposure to salt, it does facilitate residual growth from 1 h onwards. This is due to a high sink strength for solutes of the proximal part of the growth zone and its ability to accumulate solutes rapidly and at high rates.Abbreviations EDX analysis Energy-dispersive X-ray analysis - LEV Leaf elongation velocity - LVDT Linear variable differential transformer - REGR Relative elemental growth rate     

Fipronil-induced cell death in salivary glands of Rhipicephalus sanguineus (Latreille, 1806) (Acari: Ixodidae) semi-engorged females

The tick Rhipicephalus sanguineus is currently considered an urban plague. For this reason many studies are intended to find methods to control these ectoparasites. Thus, the present study analyzed the ultrastructural modifications of the salivary glands cells of semi-engorged females of R. sanguineus resulting from their exposition to Fipronil (active ingredient of Frontline®). The studied individuals were divided into four groups. Group 1 was exposed to distilled water (control) and groups 2, 3 and 4 were exposed to 1, 5 and 10 ppm of Fipronil, respectively. The salivary gland of ticks subjected to the acaricide showed accelerated process of cell death by atypical apoptosis, as well as augmented cell damages as the concentration of the chemical compound was increased. The acaricide toxicity at cellular level was demonstrated by remarkable changes of elements of the cytoskeleton and spherocrystals (extremely hard inorganic structures). However, tick defense mechanisms, such as the observed autofagic vacuoles proved the cells attempt to preserve their integrity and minimize the devastating action of this chemical compound on the salivary glands.     

Properties of condensed chromatin in barley nuclei

A method for isolation and purification of intact nuclei from barley leaves was developed and several properties of the chromatin were studied. The dense structure of the main part of the chromatin does not alter the accessibility of the DNA to nucleases. 60% of the nuclear DNA can be degraded by micrococcal endonuclease. Nevertheless the solubility of the chromatin fragments depends on the extent of nuclease digestion; solubilisation occurring only when the major part of the internucleosomal DNA was degraded (30% of digestion). Electron microscopic observations suggest that this was due to particularly dense organization of the chromatin in situ. The possible physiological meaning of some of these properties are discussed.     

Molecular imaging of cell death in vivo by a novel small molecule probe

Apoptosis has a role in many medical disorders, therefore assessment of apoptosis in vivo can be highly useful for diagnosis, follow-up and evaluation of treatment efficacy. ApoSense is a novel technology, comprising low molecular-weight probes, specifically designed for imaging of cell death in vivo. In the current study we present targeting and imaging of cell death both in vitro and in vivo, utilizing NST-732, a member of the ApoSense family, comprising a fluorophore and a fluorine atom, for both fluorescent and future positron emission tomography (PET) studies using an ¹⁸F label, respectively. In vitro, NST-732 manifested selective and rapid accumulation within various cell types undergoing apoptosis. Its uptake was blocked by caspase inhibition, and occurred from the early stages of the apoptotic process, in parallel to binding of Annexin-V, caspase activation and alterations in mitochondrial membrane potential. In vivo, NST-732 manifested selective uptake into cells undergoing cell-death in several clinically-relevant models in rodents: (i) Cell-death induced in lymphoma by irradiation; (ii) Renal ischemia/reperfusion; (iii) Cerebral stroke. Uptake of NST-732 was well-correlated with histopathological assessment of cell-death. NST-732 therefore represents a novel class of small-molecule detectors of apoptosis, with potential useful applications in imaging of the cell death process both in vitro and in vivo. Revital Aloya and Anat Shirvan are equal contribution to the paper     

The role of programmed cell death in Plasmodium–mosquito interactions

Many host–parasite interactions are regulated in part by the programmed cell death of host cells or the parasite. Here we review evidence suggesting that programmed cell death occurs during the early stages of the development of the malaria parasite in its vector. Zygotes and ookinetes of Plasmodium berghei have been shown to die by programmed cell death (apoptosis) in the midgut lumen of the vector Anopheles stephensi, or whilst developing in vitro. Several morphological markers, indicative of apoptosis, are described and evidence for the involvement of a biochemical pathway involving cysteine proteases discussed in relationship to other protozoan parasites. Malaria infection induces apoptosis in the cells of two mosquito tissues, the midgut and the follicular epithelium. Observations on cell death in both these tissues are reviewed including the role of caspases as effector molecules and the rescue of resorbing follicles resulting from inhibition of caspases. Putative signal molecules that might induce parasite and vector apoptosis are suggested including nitric oxide, reactive nitrogen intermediates, oxygen radicals and endocrine balances. Finally, we suggest that programmed cell death may play a critical role in regulation of infection by the parasite and the host, and contribute to the success or not of parasite establishment and host survival.     

Cuticular wax deposition in growing barley (<Emphasis Type="Italic">Hordeum vulgare</Emphasis>) leaves commences in relation to the point of emergence of epidermal cells from the sheaths of older leaves

In grasses, leaf cells divide and expand within the sheaths of older leaves, where the micro-environment differs from the open atmosphere. By the time epidermal cells are displaced into the atmosphere, they must have a functional cuticle to minimize uncontrolled water loss. In the present study, gas chromatography and scanning electron microscopy were used to follow cuticular wax deposition along the growing leaf three of barley (Hordeum vulgare L.). 1-Hexacosanol (C₂₆ alcohol) comprised more than 75% of extractable cuticular wax and was used as a marker for wax deposition. There was no detectable wax along the first 20 mm from the point of leaf insertion. Deposition started within the distal portion of the elongation zone (23–45 mm) and continued beyond the point of leaf emergence from the sheath of leaf two. The region where wax deposition commenced shifted towards more proximal (basal) positions when the point of leaf emergence was lowered by stripping back part of the sheath. When relative humidity in the shoot environment was elevated from 70% (standard growth conditions) to 92–96% for up to 4 days prior to analysis, wax deposition did not change significantly. The results show that cuticular waxes are deposited along the growing grass leaf independent of cell age or developmental stage. Instead, the reference point for wax deposition appears to be the point of emergence of cells into the atmosphere. The possibility of changes in relative humidity between enclosed and emerged leaf regions triggering wax deposition is discussed.     

Influence of Fe concentration in the medium on multicellular pollen grains and haploid plants induced by mannitol pretreatment in barley (Hordeum vulgare L.)

Summary. This study aims to clarify the short- and long-term effects of the iron concentration in the medium on androgenesis induced in barley by isolated microspore culture. The ultrastructural features and pectin composition of the intine wall were studied in the initial stages of androgenesis. The evolution of electron-dense iron deposits on the intine was analysed in multicellular pollen grains obtained by isolated microspore culture performed for 3, 6, and 9 days using various concentrations of FeNa₂ EDTA. Finally, the number of embryo-like structures and green plants obtained by microspore culture using different Fe concentrations was evaluated in order to estimate the optimum concentration for isolated microspore culture. Correspondence and reprints: Departamento de Bioquimica, Biologia Celular y Molecular de Plantas, Estación Experimental del Zaidin, Consejo Superior de Investigaciones Cientificas, Profesor Albareda 1, 18008 Granada, Spain.