Chasing the Origin of Viruses: Capsid-Forming Genes as a Life-Saving Preadaptation within a Community of Early Replicators

Virus capsids mediate the transfer of viral genetic information from one cell to another, thus the origin of the first viruses arguably coincides with the origin of the viral capsid. Capsid genes are evolutionarily ancient and their emergence potentially predated even the origin of first free-living cells. But does the origin of the capsid coincide with the origin of viruses, or is it possible that capsid-like functionalities emerged before the appearance of true viral entities? We set to investigate this question by using a computational simulator comprising primitive replicators and replication parasites within a compartment matrix. We observe that systems with no horizontal gene transfer between compartments collapse due to the rapidly emerging replication parasites. However, introduction of capsid-like genes that induce the movement of randomly selected genes from one compartment to another rescues life by providing the non-parasitic replicators a mean to escape their current compartments before the emergence of replication parasites. Capsid-forming genes can mediate the establishment of a stable meta-population where parasites cause only local tragedies but cannot overtake the whole community. The long-term survival of replicators is dependent on the frequency of horizontal transfer events, as systems with either too much or too little genetic exchange are doomed to succumb to replication-parasites. This study provides a possible scenario for explaining the origin of viral capsids before the emergence of genuine viruses: in the absence of other means of horizontal gene transfer between compartments, evolution of capsid-like functionalities may have been necessary for early life to prevail.     

Spatial differences in hematopoiesis but not in stem cells indicate a lack of regional patterning in definitive hematopoietic stem cells

Most tissues are patterned so that progenitors in different locations are programmed to have different properties. Stem cells from different regions of the nervous system acquire intrinsic differences in their properties as they migrate through distinct environments. Hematopoietic stem cells (HSCs) also migrate through diverse environments throughout life, raising the question of whether HSCs also acquire at least transient changes in their properties as they are exposed to diverse environments. Although we observed significant differences in hematopoiesis between the fetal liver and fetal spleen, we were not able to detect phenotypic, functional, or gene expression differences between the HSCs in these organs. Regional differences in definitive hematopoiesis are therefore not determined by regional differences between HSCs. We were also not able to detect phenotypic, functional, or gene expression differences between HSCs in different adult bone marrow compartments. Our failure to detect differences among stem cells from different regions of the hematopoietic system at the same time during development suggests that the hematopoietic system has evolved mechanisms to prevent the spatial reprogramming of HSC properties as they migrate between distinct environments.     

The KDEL receptor mediates a retrieval mechanism that contributes to quality control at the endoplasmic reticulum.

Newly synthesized proteins in the endoplasmic reticulum (ER) must fold and assemble correctly before being transported to their final cellular destination. While some misfolded or partially assembled proteins have been shown to exit the ER, they fail to escape the early secretory system entirely, because they are retrieved from post-ER compartments to the ER. We elucidate a mechanistic basis for this retrieval and characterize its contribution to ER quality control by studying the fate of the unassembled T-cell antigen receptor (TCR) alpha chain. While the steady-state distribution of TCRalpha is in the ER, inhibition of retrograde transport by COPI induces the accumulation of TCRalpha in post-ER compartments, suggesting that TCRalpha is cycling between the ER and post-ER compartments. TCRalpha associates with BiP, a KDEL protein. Disruption of the ligand-binding function of the KDEL receptor releases TCRalpha from the early secretory system to the cell surface, so that TCRalpha is no longer subject to ER degradation. Thus, our findings suggest that retrieval by the KDEL receptor contributes to mechanisms by which the ER monitors newly synthesized proteins for their proper disposal.     

The BEACH protein LvsB is localized on lysosomes and postlysosomes and limits their fusion with early endosomes

The Chediak-Higashi syndrome (CHS) is a genetic disorder caused by the loss of the BEACH protein Lyst. Impaired lysosomal function in CHS patients results in many physiological problems, including immunodeficiency, albinism and neurological problems. Dictyostelium LvsB is the ortholog of mammalian Lyst and is also important for lysosomal function. A knock-in approach was used to tag LvsB with green fluorescent protein (GFP) and express it from its single chromosomal locus. GFP-LvsB was observed on late lysosomes and postlysosomes. Loss of LvsB resulted in enlarged postlysosomes, in the abnormal localization of proton pumps on postlysosomes and their abnormal acidification. The abnormal postlysosomes in LvsB-null cells were produced by the inappropriate fusion of early endosomal compartments with postlysosomal compartments. The intermixing of compartments resulted in a delayed transit of fluid-phase marker through the endolysosomal system. These results support the model that LvsB and Lyst proteins act as negative regulators of fusion by limiting the heterotypic fusion of early endosomes with postlysosomal compartments.     

Kinetics of endosomal acidification in Dictyostelium discoideum amoebae. 31P-NMR evidence for a very acidic early endosomal compartment.

We have examined the pH of the various endosomal compartments in the amoebae of the cellular slime mould Dictyostelium discoideum. This was accomplished both by fluorescence and by in vivo 31P-NMR methods. The fluid-phase marker, fluorescein-labeled dextran, was fed to the amoebae to report the average pH of their endocytic vesicles. During the progressive loading of successive endosomal compartments, we observed an early acidification down to a minimum value of pH < or = 5.3 after 30 min at 20 degrees C followed by an increase to an average pH of 5.8 when all the endosomal compartments were loaded by the fluid-phase marker. The weak fluorescence intensity of FITC-dextran at acidic pH precluded a more detailed investigation and we checked various phosphonate compounds as potential 31P-NMR pH probes for the endosomal compartments. Two molecules, aminomethylphosphonate and 2-aminoethylphosphonate, were selected for this study because of the large amplitudes of their chemical shift variation with pH (2 and 2.5 ppm, respectively) and their acidic pKs of 5.5 and 6.3, respectively. They were only moderately toxic (IC50% approximately 10 mM) towards both the axenic growth and the differentiation program of Dictyostelium amoebae. Internalization of the two aminophosphonates occurred only through the fluid-phase pinocytosis pathway as revealed by the full inhibition of their entry with 1 mM vanadate or 7.5 mM caffeine, two previously characterized inhibitors of endocytosis in Dictyostelium. We found that in vivo 31P-NMR of amoebae suspensions incubated with the aminophosphonates allowed the detection of three distinct intracellular compartments at pH 4.3, 5.8-6.0 and 7.3. Kinetics of aminophosphonate entry were analyzed and the results allowed us to reconstruct the time course for the acidification sequence during endocytosis. The data are consistent with the hypothesis that in Dictyostelium amoebae phosphonates occupy a highly acidic early endosomal compartment (t1/2 = 18 min; pH 4.3) before reaching a less acidic late endosomal/prelysosomal compartment (pH 5.8-6.0) from where they are immediately transported to, and trapped in, the cytoplasm (pH 7.3).     

Functional symmetry of endomembranes

In higher eukaryotic cells pleiomorphic compartments composed of vacuoles, tubules and vesicles move from the endoplasmic reticulum (ER) and the plasma membrane to the cell center, operating in early biosynthetic trafficking and endocytosis, respectively. Besides transporting cargo to the Golgi apparatus and lysosomes, a major task of these compartments is to promote extensive membrane recycling. The endocytic membrane system is traditionally divided into early (sorting) endosomes, late endosomes and the endocytic recycling compartment (ERC). Recent studies on the intermediate compartment (IC) between the ER and the Golgi apparatus suggest that it also consists of peripheral ("early") and centralized ("late") structures, as well as a third component, designated here as the biosynthetic recycling compartment (BRC). We propose that the ERC and the BRC exist as long-lived "mirror compartments" at the cell center that also share the ability to expand and become mobilized during cell activation. These considerations emphasize the functional symmetry of endomembrane compartments, which provides a basis for the membrane rearrangements taking place during cell division, polarization, and differentiation.     

Regeneration of natural antibody repertoire after massive ablation of lymphoid system: robust selection mechanisms preserve antigen binding specificities

Natural Abs (NAbs) are Igs present in the serum and body fluids of healthy vertebrate animals, without any previous intentional immunization. NAbs often exhibit autoreactivity but also play an essential role in immunity, being a first line of defense against infectious microorganisms. We have previously analyzed the natural serum IgM Ab repertoire of normal mice, characterizing their reactivity with hundreds/thousands of self Ags; a significant similarity among different individuals was observed, and it was found that many reactivities of NAbs stably kept during adulthood were established early in life, implicating that period as a critical time window in the physiology of NAb repertoire selection. In the work reported here, experiments were conducted to address the role of normal lymphocyte ontogeny to the formation and stability of adult NAb repertoire. The massive destruction of the lymphoid system was promoted in adult mice with gamma-irradiation, and regeneration of hemopoietic tissues was granted by bone marrow or fetal liver inoculum. NAb repertoire regeneration was followed for 60 days after gamma-irradiation in bone marrow or fetal liver chimeric animals. The analysis of serum IgM reactivity with hundreds/thousands of self Ags showed that the NAb repertoire regenerated most of its original format after massive destruction of lymphoid compartments, characterizing autoreactive repertoire selection as a robust biological process. The data also show that regeneration of the NAb repertoire occurred similarly in fetal liver and bone marrow chimeras, although the latter animals poorly reconstituted their CD5(+) B1 cell compartment, suggesting that B1 cells are not essential for natural Ab regeneration.     

Ubiquitin initiates sorting of Golgi and plasma membrane proteins into the vacuolar degradation pathway

ABSTRACT: BACKGROUND: In yeast and mammals, many plasma membrane (PM) proteins destined for degradation are tagged with ubiquitin. These ubiquitinated proteins are internalized into clathrin-coated vesicles and are transported to early endosomal compartments. There, ubiquitinated proteins are sorted by the endosomal sorting complex required for transport (ESCRT) machinery into the intraluminal vesicles of multivesicular endosomes. Degradation of these proteins occurs after endosomes fuse with lysosomes/lytic vacuoles to release their content into the lumen. In plants, some PM proteins, which cycle between the PM and endosomal compartments, have been found to be ubiquitinated, but it is unclear whether ubiquitin is sufficient to mediate internalization and thus acts as a primary sorting signal for the endocytic pathway. To test whether plants use ubiquitin as a signal for the degradation of membrane proteins, we have translationally fused ubiquitin to different fluorescent reporters for the plasma membrane and analyzed their transport. RESULTS: Ubiquitin-tagged PM reporters localized to endosomes and to the lumen of the lytic vacuole in tobacco mesophyll protoplasts and in tobacco epidermal cells. The internalization of these reporters was significantly reduced if clathrin-mediated endocytosis was inhibited by the coexpression of a mutant of the clathrin heavy chain, the clathrin hub. Surprisingly, a ubiquitin-tagged reporter for the Golgi was also transported into the lumen of the vacuole. Vacuolar delivery of the reporters was abolished upon inhibition of the ESCRT machinery, indicating that the vacuolar delivery of these reporters occurs via the endocytic transport route. CONCLUSIONS: Ubiquitin acts as a sorting signal at different compartments in the endomembrane system to target membrane proteins into the vacuolar degradation pathway: If displayed at the PM, ubiquitin triggers internalization of PM reporters into the endocytic transport route, but it also mediates vacuolar delivery if displayed at the Golgi. In both cases, ubiquitin-tagged proteins travel via early endosomes and multivesicular bodies to the lytic vacuole. This suggests that vacuolar degradation of ubiquitinated proteins is not restricted to PM proteins but might also facilitate the turnover of membrane proteins in the early secretory pathway.     

The lipocalin Xlcpl1 expressed in the neural plate of Xenopus laevis embryos is a secreted retinaldehyde binding protein.

The cellular and structural properties and binding capabilities of a lipocalin expressed in the early neural plate of Xenopus laevis embryos and the adult choroid plexus have been investigated. It was found that this lipocalin, termed Xlcpl1, binds retinal at a nanomolar concentration, retinoic acid in the micromolar range, but does not show binding to retinol. Furthermore, this protein also binds D/L thyroxine. The Xlcpl1 cDNA was expressed in cell culture using the vaccinia virus expression system. In AtT20 cells, Xlcpl1 was secreted via the constitutive secretory pathway. We therefore assume that cpl1 binds retinaldehyde during the transport through the compartments of the secretory pathway that are considered to be the storage compartments of retinoids. Therefore, cpl1-expressing cells will secrete the precursors of active retinoids such as retinoic acid isomers. These retinoids may enter the cytosol by diffusion or receptor-controlled mechanisms, as has been shown for exogenously applied retinoids. Based on these data, it is suggested that cpl1 is an integral member of the retinoid signaling pathway and, therefore, it plays a key role in pattern formation in early embryonic development.     

Reducing feather pecking when raising laying hen chicks in aviary systems

Aviary systems for laying hens offer several advantages over battery cages. However, pecking the feathers of conspecifics remains a serious problem that negatively affects the welfare of the birds as well as the economy of a farm. From experimental studies with small groups, it has been shown that feather pecking and foraging behaviour are related and that both behaviour are influenced by early access to litter substrate. We, therefore, hypothesised, that feather pecking in aviaries can be reduced with an adequate management in the first 2 weeks of life.Each of seven pens on six commercial poultry farms, was divided into two identical compartments (matched pair design). In one of the compartments (experimental compartment) chicks were reared for the first 2 weeks of life with access to litter (wood shavings, in one case with additional straw), while the chicks in the other compartment (control) were kept on a plastic grid. Thereafter, all chicks had unrestricted access to litter and there were no differences between the two compartments neither in housing conditions nor in management procedures.Chicks in the experimental compartments spent significantly more time foraging (week 5), showed significantly less feather pecking (weeks 5 and 14) and significantly fewer birds had damaged tail feathers (weeks 5 and 14).The study demonstrates that in aviaries, under commercial conditions, early access to litter substrate has a significant effect on the development of feather pecking. In order to reduce feather pecking and to increase foraging behaviour, it is recommended that laying hen chicks raised in aviary systems do get access to litter from day 1 on.     

Receptor-mediated endocytosis of asialoglycoproteins by rat liver hepatocytes: biochemical characterization of the endosomal compartments

The endocytic compartments of the asialoglycoprotein (ASGP) pathway in rat hepatocytes were studied using a combined morphological and biochemical approach in the isolated perfused liver. Use of electron microscopic tracers and a temperature-shift protocol to synchronize ligand entry confirmed the route of ASGP internalization observed in our previous in vivo studies (1) and established conditions under which we could label the contents of successive compartments in the pathway for subcellular fractionation studies. Three endosomal compartments were demonstrated in which ASGPs appear after they enter the cell via coated pits and vesicles but before they reach their site of degradation in lysosomes. These three compartments could be distinguished by their location within the hepatocyte, by their morphological appearance in situ, and by their density in sucrose gradients. The distributions of ASGP receptors, both accessible and latent (revealed by detergent permeabilization), were also examined and compared with that of ligand during subcellular fractionation. Most accessible ASGP receptors co-distributed with conventional plasma membrane markers. However, hepatocytes contain a substantial intracellular pool of latent ASGP binding sites that exceeds the number of cell surface receptors and whose presence is not dependent on ASGP exposure. The distribution of these latent ASGP receptors on sucrose gradients (detected either immunologically or by binding assays) was coincident with that of ligand sequestered within the early endosome compartments. In addition, both early endosomes and the membrane vesicles containing latent ASGP receptors had high cholesterol content, because both shifted markedly in density upon exposure to digitonin.     

Use of fluorescent protein tags to study nuclear organization of the spliceosomal machinery in transiently transformed living plant cells

Although early studies suggested that little compartmentalization exists within the nucleus, more recent studies on metazoan systems have identified a still increasing number of specific subnuclear compartments. Some of these compartments are dynamic structures; indeed, protein and RNA-protein components can cycle between different domains. This is particularly evident for RNA processing components. In plants, lack of tools has hampered studies on nuclear compartmentalization and dynamics of RNA processing components. Here, we show that transient expression of fluorescent protein fusions of U1 and U2 small nuclear ribonucleoprotein particle (snRNP)-specific proteins U1-70K, U2B", and U2A ', nucleolar proteins Nop10 and PRH75, and serine-arginine-rich proteins in plant protoplasts results in their correct localization. Furthermore, snRNP-specific proteins also were correctly assembled into mature snRNPs. This system allowed a systematic analysis of the cellular localization of Arabidopsis serine-arginine-rich proteins, which, like their animal counterparts, localize to speckles but not to nucleoli and Cajal bodies. Finally, markers for three different nuclear compartments, namely, nucleoli, Cajal bodies, and speckles, have been established and were shown to be applicable for colocalization studies in living plant protoplasts. Thus, transient expression of proteins tagged with four different fluorescent proteins is a suitable system for studying the nuclear organization of spliceosomal proteins in living plant cells and should therefore allow studies of their dynamics as well.     

Controversies on the origin of life.

Different viewpoints, many with deep philosophical and historical roots, have shaped the scientific study of the origin of life. Some of these argue that primeval life was based on simple anaerobic microorganisms able to use a wide inventory of abiotic organic materials (i.e. a heterotrophic origin), whereas others invoke a more sophisticated organization, one that thrived on simple inorganic molecules (i.e. an autotrophic origin). While many scientists assume that life started as a self-replicative molecule, the first gene, a primitive self-catalytic metabolic network has also been proposed as a starting point. Even the emergence of the cell itself is a contentious issue: did boundaries and compartments appear early or late during life's origin? Starting with a recent definition of life, based on concepts of autonomy and open-ended evolution, it is proposed here that, firstly, organic molecules self-organized in a primordial metabolism located inside protocells. The flow of matter and energy across those early molecular systems allowed the generation of more ordered states, forming the cradle of the first genetic records. Thus, the origin of life was a process initiated within ecologically interconnected autonomous compartments that evolved into cells with hereditary and true Darwinian evolutionary capabilities. In other words, the individual existence of life preceded its historical-collective dimension.     

Human immunodeficiency virus rev-binding protein is essential for influenza a virus replication and promotes genome trafficking in late-stage infection

Influenza A virus uses cellular protein transport systems (e.g., CRM1-mediated nuclear export and Rab11-dependent recycling endosomes) for genome trafficking from the nucleus to the plasma membrane, where new virions are assembled. However, the detailed mechanisms of these events have not been completely resolved, and additional cellular factors are probably required. Here, we investigated the role of the cellular human immunodeficiency virus (HIV) Rev-binding protein (HRB), which interacts with influenza virus nuclear export protein (NEP), during the influenza virus life cycle. By using small interfering RNAs (siRNAs) and overexpression of a dominant negative HRB protein fragment, we show that cells lacking functional HRB have significantly reduced production of influenza virus progeny and that this defect results from impaired viral ribonucleoprotein (vRNP) delivery to the plasma membrane in late-stage infection. Since HRB colocalizes with influenza vRNPs early after their delivery to the cytoplasm, it may mediate a connection between the nucleocytoplasmic transport machinery and the endosomal system, thus facilitating the transfer of vRNPs from nuclear export to cytoplasmic trafficking complexes. We also found an association between NEP and HRB in the perinuclear region, suggesting that NEP may contribute to this process. Our results identify HRB as a second endosomal factor with a crucial role in influenza virus genome trafficking, suggest cooperation between unique endosomal compartments in the late steps of the influenza virus life cycle, and provide a common link between the cytoplasmic trafficking mechanisms of influenza virus and HIV.     

Regulation of Endosome Sorting by a Specific PP2A Isoform

The regulated sorting of proteins within the trans-Golgi network (TGN)/endosomal system is a key determinant of their biological activity in vivo. For example, the endoprotease furin activates of a wide range of proproteins in multiple compartments within the TGN/endosomal system. Phosphorylation of its cytosolic domain by casein kinase II (CKII) promotes the localization of furin to the TGN and early endosomes whereas dephosphorylation is required for efficient transport between these compartments (Jones, B.G., L. Thomas, S.S. Molloy, C.D. Thulin, M.D. Fry, K.A. Walsh, and G. Thomas. 1995. EMBO [Eur. Mol. Biol. Organ.] J. 14:5869–5883). Here we show that phosphorylated furin molecules internalized from the cell surface are retained in a local cycling loop between early endosomes and the plasma membrane. This cycling loop requires the phosphorylation state-dependent furin-sorting protein PACS-1, and mirrors the trafficking pathway described recently for the TGN localization of furin (Wan, L., S.S. Molloy, L. Thomas, G. Liu, Y. Xiang, S.L. Ryback, and G. Thomas. 1998. Cell. 94:205–216). We also demonstrate a novel role for protein phosphatase 2A (PP2A) in regulating protein localization in the TGN/endosomal system. Using baculovirus recombinants expressing individual PP2A subunits, we show that the dephosphorylation of furin in vitro requires heterotrimeric phosphatase containing B family regulatory subunits. The importance of this PP2A isoform in directing the routing of furin from early endosomes to the TGN was established using SV-40 small t antigen as a diagnostic tool in vivo. The role of both CKII and PP2A in controlling multiple sorting steps in the TGN/endosomal system indicates that the distribution of itinerant membrane proteins may be acutely regulated via signal transduction pathways.     

Endosomal density shift is related to a decrease in fusion capacity.

Dinitrophenol (DNP)-beta-glucuronidase and mannosylated anti-DNP IgG, which are endocytosed by the mannose receptor and delivered to lysosomes, were previously developed as probes for examination of fusion between early endosomes in a cell-free system. In this study, these probes were found to be transported by intact cells to endocytic vesicles with heavy buoyant density at different rates, as determined by Percoll gradient fractionation of cell homogenates. There was a concomitant loss of in vitro fusion activity as the ligands moved to dense compartments. In monensin-treated cells, DNP-beta-glucuronidase was retained in a light compartment corresponding to intracellular vesicles capable of fusion in vitro. Pulse-chase studies using a DNP-derivatized transferrin-alkaline phosphatase conjugate showed that a recycling ligand was always found in light intracellular vesicles that were capable of fusion to early endosomes in vitro. In contrast to cell-free systems, intact cells sequentially labeled with DNP-beta-glucuronidase and then mannosylated anti-DNP IgG showed ligand mixing in both early and late endocytic compartments. Treatment with nocodazole or colchicine did not affect the rate of DNP-beta-glucuronidase transport to heavy vesicles in intact cells, however, the extent of ligand mixing in late endosomes was decreased by microtubule disruption. Using sequentially labeled cells split into two groups, we directly compared ligand mixing in vitro to mixing by intact cells. Fusion alone does not mediate increases in vesicle density, since DNP-beta-glucuronidase/anti-DNP IgG complexes formed in vitro were found in light vesicles, while intact cells showed immune complexes predominantly in heavy vesicles. These results suggest that the density shift is an initial step in targeting to lysosomes.     

On the origins of cells: a hypothesis for the evolutionary transitions from abiotic geochemistry to chemoautotrophic prokaryotes,and from prokaryotes to nucleated cells

All life is organized as cells. Physical compartmentation from the environment and self-organization of self-contained redox reactions are the most conserved attributes of living things, hence inorganic matter with such attributes would be life's most likely forebear. We propose that life evolved in structured iron monosulphide precipitates in a seepage site hydrothermal mound at a redox, pH and temperature gradient between sulphide-rich hydrothermal fluid and iron(II)-containing waters of the Hadean ocean floor. The naturally arising, three-dimensional compartmentation observed within fossilized seepage-site metal sulphide precipitates indicates that these inorganic compartments were the precursors of cell walls and membranes found in free-living prokaryotes. The known capability of FeS and NiS to catalyse the synthesis of the acetyl-methylsulphide from carbon monoxide and methylsulphide, constituents of hydrothermal fluid, indicates that pre-biotic syntheses occurred at the inner surfaces of these metal-sulphide-walled compartments, which furthermore restrained reacted products from diffusion into the ocean, providing sufficient concentrations of reactants to forge the transition from geochemistry to biochemistry. The chemistry of what is known as the RNA-world could have taken place within these naturally forming, catalyticwalled compartments to give rise to replicating systems. Sufficient concentrations of precursors to support replication would have been synthesized in situ geochemically and biogeochemically, with FeS (and NiS) centres playing the central catalytic role. The universal ancestor we infer was not a free-living cell, but rather was confined to the naturally chemiosmotic, FeS compartments within which the synthesis of its constituents occurred. The first free-living cells are suggested to have been eubacterial and archaebacterial chemoautotrophs that emerged more than 3.8 Gyr ago from their inorganic confines. We propose that the emergence of these prokaryotic lineages from inorganic confines occurred independently, facilitated by the independent origins of membrane-lipid biosynthesis: isoprenoid ether membranes in the archaebacterial and fatty acid ester membranes in the eubacterial lineage. The eukaryotes, all of which are ancestrally heterotrophs and possess eubacterial lipids, are suggested to have arisen ca. 2 Gyr ago through symbiosis involving an autotrophic archaebacterial host and a heterotrophic eubacterial symbiont, the common ancestor of mitochondria and hydrogenosomes. The attributes shared by all prokaryotes are viewed as inheritances from their confined universal ancestor. The attributes that distinguish eubacteria and archaebacteria, yet are uniform within the groups, are viewed as relics of their phase of differentiation after divergence from the non-free-living universal ancestor and before the origin of the free-living chemoautotrophic lifestyle. The attributes shared by eukaryotes with eubacteria and archaebacteria, respectively, are viewed as inheritances via symbiosis. The attributes unique to eukaryotes are viewed as inventions specific to their lineage. The origin of the eukaryotic endomembrane system and nuclear membrane are suggested to be the fortuitous result of the expression of genes for eubacterial membrane lipid synthesis by an archaebacterial genetic apparatus in a compartment that was not fully prepared to accommodate such compounds, resulting in vesicles of eubacterial lipids that accumulated in the cytosol around their site of synthesis. Under these premises, the most ancient divide in the living world is that between eubacteria and archaebacteria, yet the steepest evolutionary grade is that between prokaryotes and eukaryotes.     

The use of floral homeotic mutants as a novel way to obtain durable resistance to insect pests

We have developed a novel strategy for the introduction of durable insect resistance in crops. This strategy was based on intervention in the natural relationship between plants and insects. For many insects, including pests such as thrips (Frankliniella occidentalis), the flower is an important factor in their life cycle, serving either as a food source or as a place for mating. The insects are attracted to the flower by scent, which is mainly produced by the petals, and by the bright colour of these floral organs. We therefore anticipated that removal or changing the identity of the petals would significantly reduce the attractiveness of the flower to thrips. To test this hypothesis, we used cucumber as a model species because most modern varieties are parthenocarpic, in which the fruit develops without fertilization. The cucumber mutant green petals, in which the petals are homeotically transformed into green sepals, was particularly useful for this study. The susceptibility of the cucumber plants to damage by thrips was determined by recording thrip numbers and by measuring leaf damage. Large differences were observed when greenhouse compartments with either wild-type or green petal mutant plants were compared. The rate of population growth of the insects on the mutant plants was significantly reduced and the leaves were almost undamaged. These results demonstrate that alterations in the structure of flowers may interfere with the life cycle of insects, providing the means for a novel and natural strategy for obtaining insect resistance.     

Microtensiometer technique for in situ measurement of soil matric potential and root water extraction from a sandy soil

The suitability of microtensiometers to measure the spatial variation of soil matric potential and its diurnal change was tested in a pot experiment with pearl millet (Pennisetum americanum [L.] Leeke) in a sandy soil as the soil dried out.The temporal and spatial resolution of this technique allowed precise measurement of soil matric potential and thus estimation of soil water extraction from different compartments as well as from the whole rooting zone. The technique also allowed the measurement of rehydration of plants at night and root water uptake rate per unit soil volume or per unit root length. The precision of determination of root water uptake depended greatly on the accuracy of the estimate of hydraulic conductivity, which was derived from a bare soil and might be different for a cropped soil owing to aggregation induced by the root system. A linear relationship between root length and water uptake was found (r²=0.82), irrespective of variation in soil water content between compartments and despite the variation in root age, xylem differentiation and suberin formation expected to exist between different compartments of the rooting zone. As the experiment was carried out in a range of soil matric potentials between –4 and –30 kPa, drought stress did not occur. Further information at lower soil matric potentials are required, to address questions such as the importance of soil resistance for water uptake, or which portion of the root system has to be stressed to induce hormonal signals to the shoot. The microtensiometer technique can be applied to soil matric potentials up to –80 kPa.     

Response of Douglas-fir and amabilis fir to split-root nutrition with inorganic and organic nitrogen

There is limited understanding of the spatial plasticity of conifer root growth in response to inorganic and organic nitrogen (N). In this study, slow-growing amabilis fir and fast-growing Douglas-fir, and slow- and fast-growing seedlots of the latter species were examined for their ability to proliferate roots preferentially in compartments of sand/peat medium enriched in organic and inorganic forms of N. In one experiment, N was supplied as 7.1 or 0.71 mM ammonium, nitrate and ammonium nitrate, and in a second experiment, N was supplied as ammonium or glycine. The seedlings’ ability to compensate for the starvation of a portion of the root system was assessed by measuring biomass of leaves, stems and roots, and foliar N concentration. Both fast- and slow-growing seedlots of Douglas-fir and slow-growing amabilis fir were able to proliferate roots in compartments of soil enriched with inorganic and organic N. In the first experiment, whole plant and root biomass was greatest when N was provided as ammonium followed by nitrate, and in the second experiment, seedling whole and root biomasses did not differ between ammonium and glycine treatments. All seedlings were able to compensate for the starvation of a portion of the root system, thus total plant biomass did not differ between split-root treatments; however, foliar N contents were lower in the 7.1/0.71 mM inorganic N split-root treatments. Foliar N concentrations were also lower in seedlings supplied with glycine.