Lack of respiratory chain complex I impairs alternative oxidase engagement and modulates redox signaling during elicitor-induced cell death in tobacco

Alternative oxidase (AOX) functions in stress resistance by preventing accumulation of reactive oxygen species (ROS), but little is known about in vivo partitioning of electron flow between AOX and the cytochrome pathway. We investigated the relationships between AOX expression and in vivo activity in Nicotiana sylvestris and the complex I-deficient CMSII mutant in response to a cell death elicitor. While a specific AOX1 isoform in the active reduced state was constitutively overexpressed in CMSII, partitioning through the alternative pathway was similar to the wild type. Lack of correlation between AOX content and activity indicates severe metabolic constraints in nonstressed mutant leaves. The bacterial elicitor harpin N(Ea) induced similar timing and extent of cell death and a twofold respiratory burst in both genotypes with little change in AOX amounts. However, partitioning to AOX was increased twofold in the wild type but remained unchanged in CMSII. Oxidative phosphorylation modeling indicated a twofold ATP increase in both genotypes. By contrast, mitochondrial superoxide dismutase activity and reduced forms of ascorbate and glutathione were higher in CMSII than in the wild type. These results demonstrate genetically programmed flexibility of plant respiratory routes and antioxidants in response to elicitors and suggest that sustained ATP production, rather than AOX activity by itself or mitochondrial ROS, might be important for in planta cell death.     

Light and oxygen are not required for harpin-induced cell death

Nicotiana sylvestris leaves challenged by the bacterial elicitor harpin N(Ea) were used as a model system in which to determine the respective roles of light, oxygen, photosynthesis, and respiration in the programmed cell death response in plants. The appearance of cell death markers, such as membrane damage, nuclear fragmentation, and induction of the stress-responsive element Tnt1, was observed in all conditions. However, the cell death process was delayed in the dark compared with the light, despite a similar accumulation of superoxide and hydrogen peroxide in the chloroplasts. In contrast, harpin-induced cell death was accelerated under very low oxygen (<0.1% O(2)) compared with air. Oxygen deprivation impaired accumulation of chloroplastic reactive oxygen species (ROS) and the induction of cytosolic antioxidant genes in both the light and the dark. It also attenuates the collapse of photosynthetic capacity and the respiratory burst driven by mitochondrial alternative oxidase activity observed in air. Since alternative oxidase is known to limit overreduction of the respiratory chain, these results strongly suggest that mitochondrial ROS accumulate in leaves elicited under low oxygen. We conclude that the harpin-induced cell death does not require ROS accumulation in the apoplast or in the chloroplasts but that mitochondrial ROS could be important in the orchestration of the cell suicide program.     

Modified uroporphyrinogen decarboxylase activity in a yeast mutant which mimics porphyria cutanea tarda.

The isolation of a new mutant Sm1 strain of yeast, Saccharomyces cerevisiae, is described: this strain was partially defective in haem formation and accumulated large amounts of Zn-porphyrins. Genetic analysis showed that the porphyrin accumulation was under the control of a single nuclear recessive mutation. Biochemical analysis showed that the main porphyrins accumulated in the cells were uroporphyrin and heptacarboxyporphyrin, mostly of the isomer-III type. The excreted porphyrins comprised mainly dehydroisocoproporphyrin. Analysis of uroporphyrinogen decarboxylase activity in the cell-free extract revealed a 70-80% decrease of activity in the mutant and showed that the relative rates of the different decarboxylation steps were modified with the mutant enzyme. A 2-3-fold increase in 5-aminolaevulinate synthase activity was measured in the mutant. The biochemical characteristics of the Sm1 mutant are very similar to those described for porphyria cutanea tarda.     

Protein-specific features of the general secretion pathway in yeast: the secretion of acid phosphatase

The major phosphate-repressible acid phosphatase (APase) of Saccharomyces cerevisiae, a cell wall glycoprotein, has been extensively used as a reporter protein to analyse successive steps in the yeast secretory pathway. In contrast to other yeast secretory proteins, APase can still be translocated into the endoplasmic reticulum (ER) even when it is made without its signal peptide. This property illustrates the permissiveness of targeting to the ER in yeast. Studies on APase-containing hybrid proteins have provided some of the evidence that specific soluble factors must interact with secretory proteins prior to their translocation across the ER membrane. A systematic analysis of mutations affecting the sequence of the APase signal peptide cleavage site demonstrated that cleavage occurs only when the last amino acid of the signal sequence is small and neutral. This was one of the first studies to verify the requirements for signal peptidase cleavage that had previously only been predicted from statistical analysis. Studies performed either with inhibitors of glycosylation or with mutant APases demonstrated the critical role of core glycosylation for APase folding, which is essential for efficient transport beyond the ER. Following the fate of particular modified APases along the secretory pathway provided insights into some general properties of the secretory apparatus and illustrated the specific requirements for a given protein during its intracellular traffic.     

Isolation and characterization of extragenic mutations affecting the expression of the uroporphyrinogen decarboxylase gene (HEM12) in Sacharomyces cerevisiae

Uroporphyrinogen decarboxylase (Uro-d; EC 4.1.1.37), the fifth enzyme in the heme biosynthetic pathway, which catalyzes the sequential decarboxylation of uroporphyrinogen to coproporphyrinogen, is encoded by the HEM12 gene in Saccharomyces cerevisiae. The HEM12 gene is transcribed into a major short mRNA and a minor longer one, approximately 1.35 and 1.55 kb, respectively, in size, and that differ in the 5 untranslated region. Uroporphyric mutants, which have no mutations in the HEM12 gene but accumulate uroporphyrinogen, a phenotype chracteristic of partial Uro-d deficiency, were investigated. Genetic analysis showed that the mutant phenotype depends on the combined action of two unlinked mutations, udt1 and either ipa1, ipa2, or ipa3. ipa1 is tightly linked to HEM12 The mutation udt1 apparently acts specifically on the HEM12 gene, and causes a six to tenfold decrease in the levels of the short HEM12 mRNA, in the -galactosidase activity of a HEM12-lacZ fusion, in immunodetectable protein and enzyme activity. But heme synthesis is normal and porphyrin accumulation was modest. The mutations ipa1, ipa2, and ipa3 had no phenotype on their own, but they caused an increase in porphyrin accumulation in a udt1 background. This multiplicity of genetic factors leading to uroporphyric yeast cells closely resembles the situation in human porphyria cutanea tarda.     

Cell sociology: A way of reconsidering the current concepts of morphogenesis

Summary Research in the field of planarian regeneration on the one hand, and a general survey of embryology on the other, throw doubt upon the reality of supra-cellular controls, which are still at the basis of all modern concepts of morphogenesis. The necessity of referring to such controls, which have never been convincingly demonstrated, is probably due to the fact that two aspects of cell behaviour have been underestimated: 1) the capacity of cells to change their individualities for a time independently of other cells; 2) the social behaviour of cells, which is the consequence of the reciprocal exchange of information. Pattern formation and pattern remodeling in normal development results from readjustments of cell populations to local or global changes. The common responses of cell populations to disturbances are enhanced mitotic activity, cessation of specific syntheses and cell migration. In the young embryo these may promptly restore the unity of the injured primordium, leading to so-called restitution; this is based on a normal sequence of further readjustments in the primordium. In older organisms the same responses give rise to cell interactions which may be the starting point for further sequential readjustments (regeneration) — in some instances these are comparable to those that originally organized the primordium in question during development. The desirability of giving up the notion of morphogenetic field is discussed.     

The role of enzyme I in the unmasking of an essential thiol of the membrane-bound enzyme II of the phosphoenolpyruvate-glucose phosphotransferase system of Escherichia coli

The membrane-bound component of the phosphotransferase system of Escherichia coli, responsible for the phosphorylative uptake of methyl-α-d-glucoside has an essential thiol group which becomes available to inactivation by thiol reagents in the presents of the phosphate-accepting sugar or when phosphoenolpyruvate synthesis is inhibited. The form resistant to the thiol reagent requires not only the absence of sugar and an intact phosphoenol-pyruvate generating system, but also an intact system generating phosphorylated Hpr which is impaired by heating of a thermosensitive enzyme I mutant.     

Cell sociology and the problem of automation in the development of pluricellular animals

The principles of automation (automatism and programming) in the unfolding of spatio-temporal patterns during animal development are deduced from experimental data reconsidered from the point of view of cell sociology. The developmental programme in the egg is not part of the genetic information but a part of the cytoplasmic information. Throughout development cells store extra-cellular information released by their neighbours in the form of cytoplasmic information. Successive determinations cannot be considered as successive reprogrammings of cells: each one consists of a selection of one specific programme from the total information previously stored. This programme specifies cell interactions in the determined population as a whole; it is very imprecise and is progressively completed during the course of further differentiation by information released by neighbouring cell populations. Complicated patterns may emerge from only two homogeneous populations involved in distinct differentiation pathways and confronting each other. Consequently the egg developmental programme provides gene effectors and specific physico-chemical conditions necessary for the starting of at least two distinct differentation pathways. Experimental data suggest that there are two components in this programme. One is a molecular machinery which starts at fertilization in the whole cytoplasm. It yields two programmes of differentiation, typically first an endodermal and then an ectodermal one. The other component of the egg developmental programme, which does not require specific information, allows the interception of the first (endodermal) programme. The application of informatics to developmental automatism is discussed in the latter part of the paper.     

Fertile Homozygous Transgenic Mice Expressing a Functional Truncated Herpes Simplex Thymidine Kinase ΔTK Gene

Dividing cells expressing the Herpes simplex type 1 thymidine kinase (TK) can be killed upon ganciclovir treatment. Likewise, conditional cell knock-out can be obtained in transgenic mice expressing a TK gene placed under the control of tissue-specific regulatory sequences. Such animals provide powerful experimental systems for assessing the functional role of specific cell populations through their time-controlled ablation. However, whatever the regulatory sequences used, a leaky toxic overexpression of TK in testis renders male TK-transgenic mice sterile and prevents the generation of homozygous TK-expressing animals. To solve this problem, we designed a truncated TK variant (TK) not expressed in the testis. We generated transgenic mice expressing TK under the control of lymphocyte-specific regulatory sequences derived from the CD4 gene. The TK protein expressed in T-lymphocytes allowed the conditional ablation of activated T-cells in vitro and in vivo. Importantly, for one transgenic line we could generate fertile homozygous mice harboring a functional TK transgene. TK should thus dramatically facilitate the development of transgenic mice expressing a conditional suicide gene.     

A Yeast t-SNARE Involved in Endocytosis

The ORF YOL018c (TLG2) of Saccharomyces cerevisiae encodes a protein that belongs to the syntaxin protein family. The proteins of this family, t-SNAREs, are present on target organelles and are thought to participate in the specific interaction between vesicles and acceptor membranes in intracellular membrane trafficking. TLG2 is not an essential gene, and its deletion does not cause defects in the secretory pathway. However, its deletion in cells lacking the vacuolar ATPase subunit Vma2p leads to loss of viability, suggesting that Tlg2p is involved in endocytosis. In tlg2Δ cells, internalization was normal for two endocytic markers, the pheromone α-factor and the plasma membrane uracil permease. In contrast, degradation of α-factor and uracil permease was delayed in tlg2Δ cells. Internalization of positively charged Nanogold shows that the endocytic pathway is perturbed in the mutant, which accumulates Nanogold in primary endocytic vesicles and shows a greatly reduced complement of early endosomes. These results strongly suggest that Tlg2p is a t-SNARE involved in early endosome biogenesis.