Competence of Immature Maize Embryos for Agrobacterium-Mediated Gene Transfer

Agrobacterium-mediated transfer of viral sequences to plant cells (agroinfection) was applied to study the susceptibility of immature maize embryos to the pathogen. The shoot apical meristem of immature embryos 10 to 20 days after pollination from four different maize genotypes was investigated for competence for agroinfection. There was a direct correlation between different morphological stages of the unwounded immature embryos and their competence for agroinfection. Agroinfection frequency was highest in the embryogenic line A188. All developmental stages tested showed Agrobacterium virulence gene-inducing activity, whereas bacteriocidal substances were produced at stages of the immature embryos competent for agroinfection. The results suggested that Agrobacterium may require differentiated tissue in the maize shoot apical meristem before wounding for successful T-DNA transfer. This requirement for the young maize embryo has implications for the possible use of Agrobacterium for maize transformation.     

Signals leading to apoptosis-dependent inhibition of neovascularization by thrombospondin-1

Thrombospondin-1 (TSP-1) is a naturally occurring inhibitor of angiogenesis that limits vessel density in normal tissues and curtails tumor growth. Here, we show that the inhibition of angiogenesis in vitro and in vivo and the induction of apoptosis by thrombospondin-1 all required the sequential activation of CD36, p59fyn, caspase-3 like proteases and p38 mitogen-activated protein kinases. We also detected increased endothelial cell apoptosis in situ at the margins of tumors in mice treated with thrombospondin-1. These results indicate that thrombospondin-1, and possibly other broad-spectrum natural inhibitors of angiogenesis, act in vivo by inducing receptor-mediated apoptosis in activated microvascular endothelial cells.     

T-DNA transfer in meristematic cells of maize provided with intracellular Agrobacterium

Agrobacterium has been established as a tool for gene delivery to most dicotyledonous plant species. However, it is not generally efficient in monocotyledonous plant species, especially not in Graminae . In maize, Agrobacterium -mediated DNA transfer has been detected but early developmental stages in the plant proved incompetent as recipients. This research tests whether the lack of competence in young immature embryos of maize could be overcome by providing Agrobacterium in the interior of the plant cell. A microinjection technique was used to target single meristematic cells and prove competence to Agrobacterium . This response is dependent on the maize plant genotype.     

Baculovirus p35 prevents developmentally programmed cell death and rescues a ced-9 mutant in the nematode Caenorhabditis elegans.

Programmed cell death, or apoptosis, occurs throughout the course of normal development in most animals and can also be elicited by a number of stimuli such as growth factor deprivation and viral infection. Certain morphological and biochemical characteristics of programmed cell death are similar among different tissues and species. During development of the nematode Caenorhabditis elegans, a single genetic pathway promotes the death of selected cells in a lineally fixed pattern. This pathway appears to be conserved among animal species. The baculovirus p35-encoding gene (p35) is an inhibitor of virus-induced apoptosis in insect cells. Here we demonstrate that expression of p35 in C. elegans prevents death of cells normally programmed to die. This suppression of developmentally programmed cell death results in appearance of extra surviving cells. Expression of p35 can rescue the embryonic lethality of a mutation in ced-9, an endogenous gene homologous to the mammalian apoptotic suppressor bcl-2, whose absence leads to ectopic cell deaths. These results support the hypothesis that viral infection can activate the same cell death pathway as is used during normal development and suggest that baculovirus p35 may act downstream or independently of ced-9 in this pathway.     

SAAT: sonication-assisted Agrobacterium-mediated transformation

Plant transformation via Agrobacterium can be limited by both host specificity and the inability of Agrobacterium to reach the proper cells in the target tissue. Described here is a new and efficient Agrobacterium-based transformation technology that overcomes these barriers and enhances DNA transfer in such diverse plant groups as dicots, monocots, and gymnosperms. This new technology, called sonication-assisted Agrobacterium-mediated transformation (SAAT), involves subjecting the plant tissue to brief periods of ultrasound in the presence of Agrobacterium. Scanning electron and light microscopy reveal that SAAT treatment produces small and uniform fissures and channels throughout the tissue allowing the Agrobacterium easy access to internal plant tissues. Unlike other transformation methods, this system has the potential to transform meristematic tissue buried under several cell layers. SAAT increases transient transformation efficiency in several different plant tissues including leaf tissue, immature cotyledons, somatic and zygotic embryos, roots, stems, shoot apices, embryogenic suspension cells and whole seedlings. A 100- to 1400-fold increase in transient - glucuronid ase expression has been demonstrated in various tissues of soybean, Ohio buckeye, cowpea, white spruce, wheat and maize. Stable transformation of both soybean and Ohio buckeye has been obtained using SAAT of embryogenic suspension culture tissues. For soybean, SAAT treatment was necessary to obtain stable transformation with this tissue     

Anti-apoptotic genes of baculoviruses

Baculoviruses possess two different classes of genes with anti-apoptptic activity: p35 and iap. The p35 gene product (P35) is able to block apoptosis induced by a variety of stimuli in phylogenetically diverse organisms. P35 has recently been shown to be capable of inhibiting the ICE/ced-3 family of cysteine proteases, a family of enzymes which are implicated in cell death and which exhibit specificity for cleavage at aspartate residues. The products of the iap genes are a distinct class of proteins containing a carboxyl ring finger and tandem duplications of a unique motif known as the BIR motif. Homologues of the baculovirus iap genes have been identified in the human genome. Both classes of baculovirus anti-apoptotic genes will continue to be important tools in defining the pathways involved in apoptosis. Since our demonstration in 1991 that a baculovirus prevents host cells from undergoing apoptosis by expressing a gene known as p35(Clem et al., 1991), the study of baculovirus-induced apoptosis and the anti-apoptotic genes they possess has led to discoveries with far-reaching implications for viral pathogenesis, human disease, and the study of cell death. It is now known that a variety of eukaryotic viruses encode genes which allow them to control cellular apoptosis. Understanding the mechanism(s) by which these viral gene products act provides fundamental insights into the pathways regulating apoptosis. In this review, we discuss the inhibition of apoptosis by baculoviruses, concentrating mainly on the nature and mechanism of action of the two classes of baculovirus genes, p35 and iap, which are able to control apoptosis in a diversity ofeukaryotes.     

Effect of heat shock on the metabolism of glutathione in maize roots

High performance liquid chromatography analyses revealed that glutathione (GSH) and cysteine are two of the major low molecular weight thiol compounds in maize root extracts. Treatment of maize roots to heat shock temperatures of 40°C resulted in a decrease of cysteine levels and an increase of GSH levels. Pulse labeling of maize roots with [³⁵S]cysteine showed that the rate of incorporation of ³⁵S into GSH or glutathione disulfide (GSSG) in heat shocked tissues was twice that in nonheat shocked tissues. In addition, extracts from heat shocked maize, barley, and soybean tissues contained an unidentified low molecular weight compound that increased from 1.2- to 8-fold within 2 hours of heat shock treatment depending on the tissue and plant involved. Our results indicate that during heat shock there is an increase in the activity of the GSH synthetizing capacity in maize root cells. The elevated synthesis of GSH may be related to the cells capacity to cope with heat stress conditions.     

Distinct protease pathways control cell shape and apoptosis in v-src-transformed quail neuroretina cells

Intracellular proteases play key roles in cell differentiation, proliferation and apoptosis. In nerve cells, little is known about their relative contribution to the pathways which control cell physiology, including cell death. Neoplastic transformation of avian neuroretina cells by p60(v-src) tyrosine kinase results in dramatic morphological changes and deregulation of apoptosis. To identify the proteases involved in the cellular response to p60(v-src), we evaluated the effect of specific inhibitors of caspases, calpains and the proteasome on cell shape changes and apoptosis induced by p60(v-src) inactivation in quail neuroretina cells transformed by tsNY68, a thermosensitive strain of Rous sarcoma virus. We found that the ubiquitin-proteasome pathway is recruited early after p60(v-src) inactivation and is critical for morphological changes, whereas caspases are essential for cell death. This study provides evidence that distinct intracellular proteases are involved in the control of the morphology and fate of v-src-transformed cells.     

Nitric oxide as a bioregulator of apoptosis

Nitric oxide (NO), synthesized from l-arginine by NO synthases, is a small, diffusible, highly reactive molecule with dichotomous regulatory roles under physiological and pathological conditions. NO can promote apoptosis (proapoptosis) in some cells, whereas it inhibits apoptosis (antiapoptosis) in other cells. This complexity is a consequence of the rate of NO production and the interaction with biological molecules such as iron, thiols, proteins, and reactive oxygen species. Long-lasting production of NO acts as a proapoptotic modulator by activating caspase family proteases through the release of mitochondrial cytochrome c into the cytosol, upregulation of p53 expression, activation of JNK/SAPK, and altering the expression of apoptosis-associated proteins including Bcl-2 family proteins. However, low or physiological concentrations of NO prevent cells from apoptosis induced by trophic factor withdrawal, Fas, TNFalpha, and lipopolysaccharide. The antiapoptotic mechanism can be understood via expression of protective genes such as heat shock proteins, Bcl-2 as well as direct inhibition of the apoptotic caspase family proteases by S-nitrosylation of the cysteine thiol. Our current understanding of the mechanisms by which NO exerts both pro- and antiapoptotic actions is discussed in this review article.     

Cloning and expression of murine IL-12.

Human IL-12 (NK cell stimulatory factor, cytotoxic lymphocyte maturation factor) is a heterodimeric cytokine that can act as a growth factor for activated human T and NK cells, enhance the lytic activity of human NK/lymphokine-activated killer cells, and stimulate the production of IFN-gamma by resting human PBMC. Because in our hands, human IL-12 did not elicit similar responses in murine lymphocytes, we have cloned and expressed the murine IL-12 subunit cDNA in order to obtain recombinant protein for murine studies. Comparison of the predicted amino acid sequences of the murine subunits with their human counterparts revealed that the p40 subunits are more highly conserved than the p35 subunits (70% vs 60% identity, respectively). The sizes of the p35 and p40 subunit mRNA were estimated to be 1.5 kb and 2.6 kb, respectively. RNA blot analysis showed that p35 mRNA was expressed in lymphoid tissues (spleen, thymus) and nonlymphoid tissues (lung, brain), whereas p40 mRNA expression was only detected in lymphoid cells. Incubation of splenocytes with pokeweed mitogen did not significantly affect p35 mRNA levels, however, it resulted in a decrease of p40 mRNA. Coexpression of the murine p35 and p40 cDNA clones in COS cells resulted in the secretion of IL-12, which was active in human and mouse T cell proliferation, murine NK cell activation, and murine IFN-gamma induction assays. Transfection of each subunit cDNA alone did not result in measurable secreted IL-12 activity. A hybrid heterodimer consisting of murine p35 and human p40 subunits retained bioactivity on murine cells; however, the combination of human p35 and murine p40 was completely inactive on murine cells. These results indicate that the observed inability of human IL-12 to act on murine cells is largely determined by the p35 subunit.     

Expression of the Arabidopsis histone H2A-1 gene correlates with susceptibility to Agrobacterium transformation

Transformation of plant cells by Agrobacterium tumefaciens involves both bacterial virulence proteins and host proteins. We have previously shown that the Arabidopsis thaliana gene H2A-1 (RAT5), which encodes histone H2A-1, is involved in T-DNA integration into the plant genome. Mutation of RAT5 results in a severely decreased frequency of transformation, whereas overexpression of RAT5 enhances the transformation frequency (Mysore et al., 2000b). We show here that the expression pattern of RAT5 correlates with plant root cells most susceptible to transformation. As opposed to a cyclin-GUS fusion gene whose expression is limited to meristematic tissues, the H2A-1 gene is expressed in many non-dividing cells. Under normal circumstances, the H2A-1 gene is expressed in the elongation zone of the root, the region that is most susceptible to Agrobacterium transformation. In addition, when roots are incubated on medium containing phytohormones, a concomitant shift in H2A-1 expression and transformation susceptibility to the root base is observed. Inoculation of root segments with a transfer-competent, but not a transformation-deficient Agrobacterium strain induces H2A-1 expression. Furthermore, pre-treatment of Arabidopsis root segments with phytohormones both induces H2A-1 expression and increases the frequency of Agrobacterium transformation. Our results suggest that the expression of the H2A-1 gene is both a marker for, and a predictor of, plant cells most susceptible to Agrobacterium transformation.     

Programmed cell death

This paper reviews data on programmed cell death (apoptosis) in animals and plants. Necrosis is a pathological scenario of cell death, which entails an inflammatory response in animal tissues. Apoptosis results in the disintegration of animal/plant cells into membrane vesicles enclosing the intracellular content, which are thereupon engulfed by adjacent or specialized cells (phagocytes) in animals. Plants lack such specialized cells, and plant cell walls prevent phagocytosis. The paper considers the main molecular mechanisms of apoptosis in animals and the pathways of activation of caspases, evolutionarily conserved cysteine proteases. A self-contained section concerns itself with the process of programmed cell death (PCD) in microorganisms including: 1) cell death in the myxomycete Dictyostelium discoideum and the parasitic flagellate Trypanosoma cruzi; 2) PCD in genetically manipulated yeast expressing the proapoptotic Bax and Bak proteins; 3) the death of a part of a prokaryotic cell population upon the depletion of nutrient resources or under stress; 4) the elimination of cells after a loss of a plasmid encoding a stable cytotoxic agent in combination with an unstable antidote; and 5) PCD in phage-infected bacterial cells.     

Annexin-mediated secretory vesicle aggregation in plants

The mechanism by which membranes fuse during vesicle-mediated secretion is of considerable importance for plant cell growth, but remains unknown. We have identified Ca²⁺-dependent phospholipid-binding proteins (annexins) from maize ( Zea mays ), that may play a part in this process. An assay for Ca²⁺-dependent binding of annexins to liposomes, revealed that the maize proteins (p23, p33 and p35) and annexins from bovine lung, bind over a similar range of Ca²⁺ concentrations. Turbidity assays further revealed that both maize and bovine annexins induced liposome aggregation and that the plant annexins were also effective at aggregating plant secretory vesicles. This aggregation occurred at levels of free Ca²⁺ similar to that required for the binding of annexins p33 and p35. We discuss the significance of these results for the plant secretory apparatus.     

Caspase enzyme activity is not essential for apoptosis during thymocyte development

Caspases, a family of cysteine proteases, are critical mediators of apoptosis. To address the importance of caspases in thymocyte development, we have generated transgenic mice that express the baculovirus protein p35, a viral caspase inhibitor, specifically in the thymus. p35 expression inhibited Fas (CD95)-, CD3-, or peptide-induced caspase activity in vitro and conferred resistance to Fas-induced apoptosis. However, p35 did not block specific peptide-induced negative selection in OT1 and HY TCR transgenic mouse models. Even the potent pharmacological caspase inhibitor zVAD-FMK (benzyloxycarbonyl-Val-Ala-Asp-fluoromethyl-ketone) could not prevent peptide-induced deletion of OT1 thymocytes, although it improved basal thymocyte survival in vitro. Moreover, the developmental block observed in rag1-/- thymocytes, which lack pre-TCR signaling, was also not rescued by p35 expression. These results indicate that caspase-independent signal transduction pathways can mediate thymocyte death during normal T cell development.     

Baculovirus inhibitor of apoptosis functions at or upstream of the apoptotic suppressor P35 to prevent programmed cell death.

Members of the inhibitor of apoptosis (iap) gene family prevent programmed cell death induced by multiple signals in diverse organisms, suggesting that they act at a conserved step in the apoptotic pathway. To investigate the molecular mechanism of iap function, we expressed epitope-tagged Op-iap, the prototype viral iap from Orgyia pseudotsugata nuclear polyhedrosis virus, by using novel baculovirus recombinants and stably transfected insect cell lines. Epitope-tagged Op-iap blocked both virus- and UV radiation-induced apoptosis. With or without apoptotic stimuli, Op-IAP protein (31 kDa) cofractionated with cellular membranes and the cytosol, suggesting a cytoplasmic site of action. To identify the step(s) at which Op-iap blocks apoptosis, we monitored the effect of Op-iap expression on in vivo activation of the insect CED-3/ICE death proteases (caspases). Op-iap prevented in vivo caspase-mediated cleavage of the baculovirus substrate inhibitor P35 and blocked caspase activity upon viral infection or UV irradiation. However, unlike the stoichiometric inhibitor P35, Op-IAP failed to affect activated caspase as determined by in vitro protease assays. These findings provide the first biochemical evidence that Op-iap blocks activation of the host caspase or inhibits its activity by a mechanism distinct from P35. Moreover, as suggested by the capacity of Op-iap to block apoptosis induced by diverse signals, including virus infection and UV radiation, iap functions at a central point at or upstream from steps involving the death proteases.     

蛋白激酶组在玉米叶片ABA和H202诱导抗氧化防护中的作用

蛋白磷酸化在植物细胞脱落酸（ABA）介导的信号转导中起重要作用。然而,很多参与ABA信号途径的蛋白元件仍不清楚。使用改进的体外激酶试验方法的研究结果表明,在玉米叶片中,ABA和H2O2能够快速活化蛋白激酶总活性和ca^2＋依赖型蛋白激酶总活性;ABA诱导的蛋白激酶总活性增加可以被活性氧的抑制剂和清除剂抑制,蛋白激酶抑制剂不仅可以降低ABA和H2O2诱导的激酶活性增加,而且也可以弱化它们对抗氧化防护酶活性的诱导作用;ABA和H2O2引发的蛋白磷酸化作用显著居先于它们诱导的抗氧化防护作用。使用凝胶激酶试验方法进行研究发现,一组分子量分别为66kDa,52kDa,49kDa和35kDa的蛋白激酶可能介导了ABA和H2O2诱导的抗氧化防护反应,并且66kDa和49kDa的蛋白激酶可能在ROS的下游起作用,而52kDa和35kDa的蛋白激酶可能在ABA和ROS的下游起作用。     

Mitochondria,the killer organelles and their weapons

Apoptosis is a cell-autonomous mode of death that is activated to eradicate superfluous, damaged, mutated, or aged cells. In addition to their role as the cell's powerhouse, mitochondria play a central role in the control of apoptosis. Thus, numerous pro-apoptotic molecules act on mitochondria and provoke the permeabilization of mitochondrial membranes. Soluble proteins contained in the mitochondrial intermembrane space are released through the outer membrane and participate in the organized destruction of the cell. Several among these lethal proteins can activate caspases, a class of cysteine proteases specifically activated in apoptosis, whereas others act in a caspase-independent fashion, by acting as nucleases (e.g., endonuclease G), nuclease activators (e.g., apoptosis-inducing factor), or serine proteases (e.g., Omi/HtrA2). In addition, mitochondria can generate reactive oxygen species, following uncoupling and/or inhibition of the respiratory chain. The diversity of mitochondrial factors participating in apoptosis emphasizes the central role of these organelles in apoptosis control and unravels novel mechanisms of cell death execution.     

Two faces of p53: aging and tumor suppression

The p53 tumor suppressor protein, often termed guardian of the genome, integrates diverse physiological signals in mammalian cells. In response to stress signals, perhaps the best studied of which is the response to DNA damage, p53 becomes functionally active and triggers either a transient cell cycle arrest, cell death (apoptosis) or permanent cell cycle arrest (cellular senescence). Both apoptosis and cellular senescence are potent tumor suppressor mechanisms that irreversibly prevent damaged cells from undergoing neoplastic transformation. However, both processes can also deplete renewable tissues of proliferation-competent progenitor or stem cells. Such depletion, in turn, can compromise the structure and function of tissues, which is a hallmark of aging. Moreover, whereas apoptotic cells are by definition eliminated from tissues, senescent cells can persist, acquire altered functions, and thus alter tissue microenvironments in ways that can promote both cancer and aging phenotypes. Recent evidence suggests that increased p53 activity can, at least under some circumstances, promote organismal aging. Here, we discuss the role of p53 as a key regulator of the DNA damage responses, and discuss how p53 integrates the outcome of the DNA damage response to optimally balance tumor suppression and longevity.     

Caspase- and serine protease-dependent apoptosis by the death domain of FADD in normal epithelial cells

The adapter protein FADD consists of two protein interaction domains: a death domain and a death effector domain. The death domain binds to activated death receptors such as Fas, whereas the death effector domain binds to procaspase 8. An FADD mutant, which consists of only the death domain (FADD-DD), inhibits death receptor-induced apoptosis. FADD-DD can also activate a mechanistically distinct, cell type-specific apoptotic pathway that kills normal but not cancerous prostate epithelial cells. Here, we show that this apoptosis occurs through activation of caspases 9, 3, 6, and 7 and a serine protease. Simultaneous inhibition of caspases and serine proteases prevents FADD-DD-induced death. Inhibition of either pathway alone does not prevent cell death but does affect the morphology of the dying cells. Normal prostate epithelial cells require both the caspase and serine protease inhibitors to efficiently prevent apoptosis in response to TRAIL. In contrast, the serine protease inhibitor does not affect TRAIL-induced death in prostate tumor cells suggesting that the FADD-DD-dependent pathway can be activated by TRAIL. This apoptosis pathway is activated in a cell type-specific manner that is defective in cancer cells, suggesting that this pathway may be targeted during cancer development.