Quartz-Seq: a highly reproducible and sensitive single-cell RNA sequencing method,reveals non-genetic gene-expression heterogeneity

Development of a highly reproducible and sensitive single-cell RNA sequencing (RNA-seq) method would facilitate the understanding of the biological roles and underlying mechanisms of non-genetic cellular heterogeneity. In this study, we report a novel single-cell RNA-seq method called Quartz-Seq that has a simpler protocol and higher reproducibility and sensitivity than existing methods. We show that single-cell Quartz-Seq can quantitatively detect various kinds of non-genetic cellular heterogeneity, and can detect different cell types and different cell-cycle phases of a single cell type. Moreover, this method can comprehensively reveal gene-expression heterogeneity between single cells of the same cell type in the same cell-cycle phase.     

Systems Mapping for Hematopoietic Progenitor Cell Heterogeneity

Cells with the same genotype growing under the same conditions can show different phenotypes, which is known as “population heterogeneity”. The heterogeneity of hematopoietic progenitor cells has an effect on their differentiation potential and lineage choices. However, the genetic mechanisms governing population heterogeneity remain unclear. Here, we present a statistical model for mapping the quantitative trait locus (QTL) that affects hematopoietic cell heterogeneity. This strategy, termed systems mapping, integrates a system of differential equations into the framework for systems mapping, allowing hypotheses regarding the interplay between genetic actions and cell heterogeneity to be tested. A simulation approach based on cell heterogeneity dynamics has been designed to test the statistical properties of the model. This model not only considers the traditional QTLs, but also indicates the methylated QTLs that can illustrate non-genetic individual differences. It has significant implications for probing the molecular, genetic and epigenetic mechanisms of hematopoietic progenitor cell heterogeneity.     

Differential Expression Levels of Integrin α6 Enable the Selective Identification and Isolation of Atrial and Ventricular Cardiomyocytes

Rationale

Central questions such as cardiomyocyte subtype emergence during cardiogenesis or the availability of cardiomyocyte subtypes for cell replacement therapy require selective identification and purification of atrial and ventricular cardiomyocytes. However, current methodologies do not allow for a transgene-free selective isolation of atrial or ventricular cardiomyocytes due to the lack of subtype specific cell surface markers.

Methods and Results

In order to develop cell surface marker-based isolation procedures for cardiomyocyte subtypes, we performed an antibody-based screening on embryonic mouse hearts. Our data indicate that atrial and ventricular cardiomyocytes are characterized by differential expression of integrin α6 (ITGA6) throughout development and in the adult heart. We discovered that the expression level of this surface marker correlates with the intracellular subtype-specific expression of MLC-2a and MLC-2v on the single cell level and thereby enables the discrimination of cardiomyocyte subtypes by flow cytometry. Based on the differential expression of ITGA6 in atria and ventricles during cardiogenesis, we developed purification protocols for atrial and ventricular cardiomyocytes from mouse hearts. Atrial and ventricular identities of sorted cells were confirmed by expression profiling and patch clamp analysis.

Conclusion

Here, we introduce a non-genetic, antibody-based approach to specifically isolate highly pure and viable atrial and ventricular cardiomyocytes from mouse hearts of various developmental stages. This will facilitate in-depth characterization of the individual cellular subsets and support translational research applications.     

Evaluation of the Efficacy & Biochemical Mechanism of Cell Death Induction by Piper longum Extract Selectively in In-Vitro and In-Vivo Models of Human Cancer Cells

Background

Currently chemotherapy is limited mostly to genotoxic drugs that are associated with severe side effects due to non-selective targeting of normal tissue. Natural products play a significant role in the development of most chemotherapeutic agents, with 74.8% of all available chemotherapy being derived from natural products.

Objective

To scientifically assess and validate the anticancer potential of an ethanolic extract of the fruit of the Long pepper (PLX), a plant of the piperaceae family that has been used in traditional medicine, especially Ayurveda and investigate the anticancer mechanism of action of PLX against cancer cells.

Materials & Methods

Following treatment with ethanolic long pepper extract, cell viability was assessed using a water-soluble tetrazolium salt; apoptosis induction was observed following nuclear staining by Hoechst, binding of annexin V to the externalized phosphatidyl serine and phase contrast microscopy. Image-based cytometry was used to detect the effect of long pepper extract on the production of reactive oxygen species and the dissipation of the mitochondrial membrane potential following Tetramethylrhodamine or 5,5,6,6′-tetrachloro-1,1′,3,3′-tetraethylbenzimidazolylcarbocyanine chloride staining (JC-1). Assessment of PLX in-vivo was carried out using Balb/C mice (toxicity) and CD-1 nu/nu immunocompromised mice (efficacy). HPLC analysis enabled detection of some primary compounds present within our long pepper extract.

Results

Our results indicated that an ethanolic long pepper extract selectively induces caspase-independent apoptosis in cancer cells, without affecting non-cancerous cells, by targeting the mitochondria, leading to dissipation of the mitochondrial membrane potential and increase in ROS production. Release of the AIF and endonuclease G from isolated mitochondria confirms the mitochondria as a potential target of long pepper. The efficacy of PLX in in-vivo studies indicates that oral administration is able to halt the growth of colon cancer tumors in immunocompromised mice, with no associated toxicity. These results demonstrate the potentially safe and non-toxic alternative that is long pepper extract for cancer therapy.     

Cigarette Experimentation and the Population Attributable Fraction for Associated Genetic and Non-Genetic Risk Factors

Background

We, and others, have shown that experimenting with cigarettes is a function of both non-genetic and genetic factors. In this analysis we ask: how much of the total risk of experimenting with cigarettes, among those who had not experimented with cigarettes when they enrolled in a prospective cohort, is attributable to genetic factors and to non-genetic factors?

Methods

Participants (N = 1,118 Mexican origin youth), recruited from a large population-based cohort study in Houston, Texas, provided prospective data on cigarette experimentation over three years. Non-genetic data were elicited twice – baseline and follow-up. Participants were genotyped for 672 functional and tagging variants in the dopamine, serotonin and opioid pathways.

Results

In the overall model, the adjusted combined non-genetic PAF was 71.2% and the adjusted combined genetic PAF was 58.5%. Among committed never smokers the adjusted combined non-genetic PAF was 67.0% and the adjusted combined genetic PAF was 53.5%. However, among cognitively susceptible youth, the adjusted combined non-genetic PAF was 52.0% and the adjusted combined genetic PAF was 68.4%.

Conclusions

Our results suggest there may be differences in genotypes between youth who think they will try cigarettes in the future compared to their peers who think they will not and underscore the possibility that the relative influence of genetic vs. non-genetic factors on the uptake of smoking may vary between these two groups of youth.

Impact

A clearer understanding of the relative role of genetic vs. non-genetic factors in the uptake of smoking may have implications for the design of prevention programs.     

Cell cycle- and age-dependent activation of Sod1p drives the formation of stress resistant cell subpopulations within clonal yeast cultures

Phenotypic heterogeneity describes non-genetic variation that exists between individual cells within isogenic populations. The basis for such heterogeneity is not well understood, but it is evident in a wide range of cellular functions and phenotypes and may be fundamental to the fitness of microorganisms. Here we use a suite of novel assays applied to yeast, to provide an explanation for the classic example of heterogeneous resistance to stress (copper). Cell cycle stage and replicative cell age, but not mitochondrial content, were found to be principal parameters underpinning differential Cu resistance: cell cycle-synchronized cells had relatively uniform Cu resistances, and replicative cell-age profiles differed markedly in sorted Cu-resistant and Cu-sensitive subpopulations. From a range of potential Cu-sensitive mutants, cup1Delta cells lacking Cu-metallothionein, and particularly sod1Delta cells lacking Cu, Zn-superoxide dismutase, exhibited diminished heterogeneity. Furthermore, age-dependent Cu resistance was largely abolished in cup1Delta and sod1Delta cells, whereas cell cycle-dependent Cu resistance was suppressed in sod1Delta cells. Sod1p activity oscillated approximately fivefold during the cell cycle, with peak activity coinciding with peak Cu-resistance. Thus, phenotypic heterogeneity in copper resistance is not stochastic but is driven by the progression of individual cells through the cell cycle and ageing, and is primarily dependent on only Sod1p, out of several gene products that can influence the averaged phenotype. We propose that such heterogeneity provides an important insurance mechanism for organisms; creating subpopulations that are pre-equipped for varied activities as needs may arise (e.g. when faced with stress), but without the permanent metabolic costs of constitutive expression.     

Discovery of Power-Law Growth in the Self-Renewal of Heterogeneous Glioma Stem Cell Populations

Background

Accumulating evidence indicates that cancer stem cells (CSCs) drive tumorigenesis. This suggests that CSCs should make ideal therapeutic targets. However, because CSC populations in tumors appear heterogeneous, it remains unclear how CSCs might be effectively targeted. To investigate the mechanisms by which CSC populations maintain heterogeneity during self-renewal, we established a glioma sphere (GS) forming model, to generate a population in which glioma stem cells (GSCs) become enriched. We hypothesized, based on the clonal evolution concept, that with each passage in culture, heterogeneous clonal sublines of GSs are generated that progressively show increased proliferative ability.

Methodology/Principal Findings

To test this hypothesis, we determined whether, with each passage, glioma neurosphere culture generated from four different glioma cell lines become progressively proliferative (i.e., enriched in large spheres). Rather than monitoring self-renewal, we measured heterogeneity based on neurosphere clone sizes (#cells/clone). Log-log plots of distributions of clone sizes yielded a good fit (r>0.90) to a straight line (log(% total clones) = k*log(#cells/clone)) indicating that the system follows a power-law (y = x^k) with a specific degree exponent (k = −1.42). Repeated passaging of the total GS population showed that the same power-law was maintained over six passages (CV = −1.01 to −1.17). Surprisingly, passage of either isolated small or large subclones generated fully heterogeneous populations that retained the original power-law-dependent heterogeneity. The anti-GSC agent Temozolomide, which is well known as a standard therapy for glioblastoma multiforme (GBM), suppressed the self-renewal of clones, but it never disrupted the power-law behavior of a GS population.

Conclusions/Significance

Although the data above did not support the stated hypothesis, they did strongly suggest a novel mechanism that underlies CSC heterogeneity. They indicate that power-law growth governs the self-renewal of heterogeneous glioma stem cell populations. That the data always fit a power-law suggests that: (i) clone sizes follow continuous, non-random, and scale-free hierarchy; (ii) precise biologic rules that reflect self-organizing emergent behaviors govern the generation of neurospheres. That the power-law behavior and the original GS heterogeneity are maintained over multiple passages indicates that these rules are invariant. These self-organizing mechanisms very likely underlie tumor heterogeneity during tumor growth. Discovery of this power-law behavior provides a mechanism that could be targeted in the development of new, more effective, anti-cancer agents.     

Regulation of Ethylene Biosynthesis Under Salt Stress in Red Pepper (<Emphasis Type="Italic">Capsicum annuum</Emphasis> L.) by 1-Aminocyclopropane-1-Carboxylic Acid (ACC) Deaminase-producing Halotolerant Bacteria

The present study was carried out to understand the mechanism of salt stress amelioration in red pepper plants by inoculation of 1-aminocyclopropane-1-carboxylic acid (ACC) deaminase-producing halotolerant bacteria. In general, ethylene production, ACC concentration, ACC synthase (ACS), and ACC oxidase (ACO) enzyme activities increased with increasing levels of salt stress. Treatment with halotolerant bacteria reduced ethylene production by 47–64%, ACC concentration by 47–55% and ACO activity by 18–19% in salt-stressed (150 mmol NaCl) red pepper seedlings compared to uninoculated controls. ACS activity was lower in red pepper seedlings treated with Bacillus aryabhattai RS341 but higher in seedlings treated with Brevibacterium epidermidis RS15 (44%) and Micrococcus yunnanensis RS222 (23%) under salt-stressed conditions as compared to uninoculated controls. A significant increase was recorded in red pepper plant growth under salt stress when treated with ACC deaminase-producing halotolerant bacteria as compared to uninoculated controls. The results of this study collectively suggest that salt stress enhanced ethylene production by increasing enzyme activities of the ethylene biosynthetic pathway. Inoculation with ACC deaminase-producing halotolerant bacteria plays an important role in ethylene metabolism, particularly by reducing the ACC concentration, although a direct effect on reducing ACO activity was also observed. It is suggested that growth promotion in inoculated red pepper plants under inhibitory levels of salt stress is due to ACC deaminase activity present in the halotolerant bacteria.     

Systemic Acquired Resistance of Pepper to Microbial Pathogens

To cope with the challenge of pathogens, plants have evolved a wide variety of resistance mechanisms that rely both on constitutive and on inducible defences. Systemic acquired resistance (SAR), a form of inducible resistance that occurs following an earlier localized exposure to a pathogen, provides a long‐lasting systemic immunity against a wide range of pathogens in plants. The great benefits of SAR lead to its practical use in agriculture for plant disease management. Pepper (Capsicum annuum) is one of the economically important crops growing worldwide, and in this review, we summarize the scientific research‐based studies of SAR in pepper during the past decades. Effects of various exogenous inducers of SAR, such as salicylic acid, DL‐β‐amino‐n‐butyric acid, benzothiadiazols and avirulent pathogens on pepper plants have been extensively investigated by different research groups. Biochemical and molecular studies of SAR phenomena also revealed the involvement of radical burst, cell death, endogenous hormonal signalling and defence‐related gene expression during SAR establishment in pepper. New knowledge and understanding emerging from the pepper SAR studies will allow the development of novel approaches to enhance the durable resistance of pepper to pathogens, thereby helping to secure the future supply of safe and nutritious pepper worldwide.     

The Information Value of Non-Genetic Inheritance in Plants and Animals

Parents influence the development of their offspring in many ways beyond the transmission of DNA. This includes transfer of epigenetic states, nutrients, antibodies and hormones, and behavioural interactions after birth. While the evolutionary consequences of such non-genetic inheritance are increasingly well understood, less is known about how inheritance mechanisms evolve. Here, we present a simple but versatile model to explore the adaptive evolution of non-genetic inheritance. Our model is based on a switch mechanism that produces alternative phenotypes in response to different inputs, including genes and non-genetic factors transmitted from parents and the environment experienced during development. This framework shows how genetic and non-genetic inheritance mechanisms and environmental conditions can act as cues by carrying correlational information about future selective conditions. Differential use of these cues is manifested as different degrees of genetic, parental or environmental morph determination. We use this framework to evaluate the conditions favouring non-genetic inheritance, as opposed to genetic determination of phenotype or within-generation plasticity, by applying it to two putative examples of adaptive non-genetic inheritance: maternal effects on seed germination in plants and transgenerational phase shift in desert locusts. Our simulation models show how the adaptive value of non-genetic inheritance depends on its mechanism, the pace of environmental change, and life history characteristics.     

Heterogeneity of eukaryotic replicons, replicon clusters, and replication foci

According to the current paradigm, replication foci are discrete sites in the interphase nucleus where assemblies of DNA replication enzymes simultaneously elongate the replication forks of 10–100 adjacent replicons (each ∼100 kbp). Here we review new results and provide alternative interpretations for old results to show that the current paradigm is in need of further development. In particular, many replicons are larger than previously thought – so large that their complete replication takes much longer (several hours) than the measured average time to complete replication at individual foci (45–60 min). In addition to this large heterogeneity in replicon size, it is now apparent that there is also a corresponding heterogeneity in the size and intensity of individual replication foci. An important property of all replication foci is that they are stable structures that persist, with constant dimensions, during all cell cycle stages including mitosis, and therefore likely represent a fundamental unit of chromatin organization. With this in mind, we present a modified model of replication foci in which many of the foci are composed of clusters of small replicons as previously proposed, but the size and number of replicons per focus is extremely heterogeneous, and a significant proportion of foci are composed of single large replicons. We further speculate that very large replicons may extend over two or more individual foci and that this organization may be important in regulating the replication of such large replicons as the cell proceeds through S-phase. Received: 16 August 1999 / Accepted: 17 August 1999     

A COSII genetic map of the pepper genome provides a detailed picture of synteny with tomato and new insights into recent chromosome evolution in the genus Capsicum

We report herein the development of a pepper genetic linkage map which comprises 299 orthologous markers between the pepper and tomato genomes (including 263 conserved ortholog set II or COSII markers). The expected position of additional 288 COSII markers was inferred in the pepper map via pepper–tomato synteny, bringing the total orthologous markers in the pepper genome to 587. While pepper maps have been previously reported, this is the first complete map in the sense that all markers could be placed in 12 linkage groups corresponding to the 12 chromosomes. The map presented herein is relevant to the genomes of cultivated C. annuum and wild C. annuum (as well as related Capsicum species) which differ by a reciprocal chromosome translocation. This map is also unique in that it is largely based on COSII markers, which permits the inference of a detailed syntenic relationship between the pepper and tomato genomes—shedding new light on chromosome evolution in the Solanaceae. Since divergence from their last common ancestor is approximately 20 million years ago, the two genomes have become differentiated by a minimum number of 19 inversions and 6 chromosome translocations, as well as numerous putative single gene transpositions. Nevertheless, the two genomes share 35 conserved syntenic segments (CSSs) within which gene/marker order is well preserved. The high resolution COSII synteny map described herein provides a platform for cross-reference of genetic and genomic information (including the tomato genome sequence) between pepper and tomato and therefore will facilitate both applied and basic research in pepper. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Non-genetic adaptation to temperature and salinity

Discussion and summary 1. Our present information on non-genetic adaptation of intact aquatic organisms to temperature and salinity does not yet provide a sufficient platform for a detailed analysis. Only a few of the publications available deal with non-genetic adaptation exclusively; many are primarily devoted to other topics. The mechanisms of most types of adjustments appear to be rather complex and are not yet well understood. The net result of non-genetic adaptation is compensation for adversive aspects in a changing environment.2. Non-genetic adaptation may involve quantitative changes in lethal limits, activity, metabolism, reproduction and other functions as well as in body dimensions, architecture of organs and cells, cell number per organ and in the quantity and activity of enzymes. It practically involves all levels of organismic function and structure. Non-genetic adaptation is not the result of a single process but represents a syndrome. The capacity for non-genetic adaptation depends on the genetic background of the organism involved; it may be different in different ontogenetic stages, such as egg, larva and adult, and may bear relations to metamorphosis and reproduction. There appears to be some evidence that non-genetic adaptations which have been acquired during the most sensitive phase of an individual's life cycle may be transferred to the next generation as non-genetic transmission (e. g.Prosser 1958).3. There is urgent need for carefully conducted long-term experiments. Much of our present knowledge on non-genetic adaptation has been obtained from organisms kept under inadequate conditions; numerous experiments seem to have been conducted on sick or dying specimens. Even the small amount of information available at this time has therefore to be evaluated with some critical skepticism. Poor conditions and poor health are dangerous prerequisities for the analysis of such a complex and subtle process as is non-genetic adaptation.4. Assessment of quantitative aspects of non-genetic adaptation requires distinction between its amount, stability and velocity. To illustrate this point, let us consider a euryplastic organism with a considerable capacity for non-genetic adaptation. In such an organism the amount of non-genetic adaptation tends to reach the highest values during early ontogeny and thereafter to decrease gradually with increasing age of the individual. The maximum amount of a given acclimation may only be attainable in individuals born and raised in the test environment. The amount may be expressed in percentage perfection. The perfection of a nongenetic adaptation is 100 per cent in the rare case of an ideal or perfect acclimation, i. e. if the steady-state performance following a significant change in temperature or salinity goes back to its original level after stabilization. In most cases the percentage perfection is much smaller. Thus in the crabPachigrapsus crassipes perfection of acclimation to a seasonal range of about 10°C (Southern California) was calculated byRoberts (1957) from rate-temperature curves for individuals acclimated to experimental temperatures to be about 30 per cent. The degree of stability of a non-genetic adaptation, too, seems to decrease with increasing age: adjustments during early ontogenetic development tend to be more stable than those performed during later periods of ontogeny and may even be — at least in part — irreversible throughout the rest of the life of the individual concerned. Examples areCrangon crangon (Broekema 1941),Gammarus duebeni (Kinne 1953, 1958b),Lebistes reticulatus (Gibson 1954,Fry 1957,Tsukuda &Katayama 1957,Tsukuda 1960),Cyprinodon macularius (Kinne 1962). Reversible acclimations need reinforcement if they are to be maintained. The velocity of non-genetic adaptation tends to increase with increasing rates of metabolism. In the fishCyprinodon macularius, for example, speed of acclimation increases with temperature and seems to be proportional to growth rate: fast-growing fish adapt faster than slow-growing ones (Kinne 1960, 1962).5. Most authors have considered non-genetic adaptations to a single environmental factor, namely either to temperature or salinity. Organisms, however, react to their total environment rather than to single entities. It is therefore of particular importance to study the combined effects of two or more components of the environment. Very little is presently known about the combined effects of temperature and salinity on the process of non-genetic adaptation (e. g.Dehnel 1960,Todd &Dehnel 1960,Matutani 1962,Alderdice 1963,Kinne 1963b, 1964a, b).McLeese (1956) analyzed the combined effects of temperature, salinity and oxygen on the survival rates of American lobsters (Fig. 8) (see alsoAlderdice 1963), and at the present Symposium,Roberts (1964) reported that the perfection of thermal acclimation of respiration in sunfishLepomis gibbosus becomes a function of day length above temperature of about 10°C.6. There appears to be some evidence that acclimation to one factor, say salinity, proceeds at different rates and at different efficiencies under different levels of other simultaneous acclimations, for example, to temperature or oxygen (Kinne 1964a, b). Furthermore, inharmonious interrelations between one functional or structural adaptate relative to another may be a fundamental way of limiting the total resulting amount of non-genetic adaptation. The maximum amount of acclimation to a given temperature is presumably only attainable at normal or near optimum salinities, and, conversely, maximum acclimation to salinity is presumably only possible under corresponding temperature conditions.7. Very little is known about the process of de-adaptation. Does the process of de-acclimation display a similar or a different time course than the respective acclimation? Can de-acclimation from one factor, such as temperature, be initiated or hastened by applying a new stress, such as extreme salinity? De-acclimation may involve active changes and not just a cessation of a given non-genetic adaptation. Thus upon return from high altitude to sea level, erythropoiesis not only stops, but erythrocyte destruction is accelerated (Merino 1950). Apparently, acclimation and de-acclimation are two opposed processes in competition, reaching equilibrium only under constant environmental conditions.8. The information presented in this paper pertains to reactions of intact, whole individuals. Can we expect cells, tissues or organs removed from multi-cellular animals to preserve and display the full amount of a given non-genetic adaptation acquired in the intact organism? Presumably not, if a substantial part of that acclimation is based on adjustments in organismic integration. But even in other cases, removed cells or organs may often tend to lose part or all of the acclimation acquired due to damages caused by operation procedures. Another important question is whether or not there exists a relationship between the amount of acclimation retained in isolated cells and (a) the level of organismic organization of the test organism (e. g. in the series plant, protozoan, crustacean, fish), or (b) the degree of disturbance caused by the removal of these cells.9. The amount of cellular acclimation to a given environmental situation may very well be different in different tissues or organs. Thus non-genetic adaptation to changes in salinity may express itself in cells of epidermis, gill or gut rather than in muscle or nerve cells. InCordylophora caspia, for example, acclimation to different salinities results in considerable adjustments in the cells of tentacles, hydranth body and neck, while those of the hydrocaulus and stolons remain practically unaffected (Kinne 1958a), and in male rats, cold acclimation causes a remarkable increase in the amount of brown fat, while other tissues do not seem to show such intensive modifications (Smith 1964).

---

Nichtgenetische Adaptation an Temperatur und Salzgehalt

Kurzfassung Das Phänomen der Adaptation wird aus der Perspektive der Ökologie interpretiert und der Begriff Adaptation definiert als Neueinstellung lebender Systeme im Anschluß an Veränderungen in den Intensitätsmustern von Umweltfaktoren, welche letztlich zu einer relativen Erhöhung der Überlebens-, Vermehrungs- oder Konkurrenzkapazität führt und somit objektiv meßbare potentielle Vorteile im existenzökologischen Sinne beinhaltet. Nichtgenetische Adaptationen (Synonyma: Akklimatisation, Akklimatisierung) vermögen erhebliche Veränderungen in der quantitativen Biologie des Stoffwechsels herbeizuführen. Im zeitlichen Ablauf des Akklimatisationsgeschehens werden drei Phasen unterschieden: Simultanreaktion, Stabilisierung und neuer stationärer Zustand. Eine Simultanreaktion auf plötzliche Umweltveränderungen spielt sich ab in Sekunden, Minuten oder Stunden. Der Vorgang der Stabilisierung dauert (bei marinen Wirbellosen und Fischen) gewöhnlich Tage oder Wochen, wobei oftmals mehr als die Hälfte des Endanpassungsvolumens im allerersten Abschnitt der Stabilisierungsphase bewältigt wird. Die mit dem neuen stationären Zustand verbundenen quantitativen Unterschiede gegenüber der Ausgangssituation werden insbesondere am Beispiel der oberen und unteren Letalgrenzen und der Stoffwechselintensität erläutert. Nichtgenetische Adaptationen können sich aber auch auf andere Funktionen erstrecken, wie etwa Bewegungsaktivität, Vermehrung oder Verhalten und auf strukturelle Bereiche (Körperdimensionen, Organ- und Zellarchitektur, Zellzahl pro Organ etc.). Eine eingehende Beurteilung der quantitativen Aspekte nichtgenetischer Adaptationen erfordert eine Differenzierung zwischen Gesamtvolumen (amount; percentage perfection), Stabilität und Rate. Das Gesamtvolumen erreicht häufig Maximalwerte während der frühen Ontogenie und nimmt danach mit zunehmendem Alter ab. Ähnliches gilt für die Stabilität: Während der frühen Ontogenie erworbene Akklimatisationen erweisen sich häufig als besonders stabil und können im Verlauf des späteren Individualdaseins sogar partiell irreversibel sein. Die Akklimatisationsrate steigt gewöhnlich mit zunehmender Stoffwechselintensität. Vermutlich vollzieht sich eine nichtgenetische Adaptation gegenüber einem Faktor, etwa Salzgehalt, mit unterschiedlicher Geschwindigkeit und unterschiedlichem Nutzeffekt bei verschiedenen Intensitätsmustern eines gleichzeitig einwirkenden zweiten oder dritten Faktors (Temperatur, Sauerstoffgehalt).

---

---

This paper is dedicated to Prof. Dr. Wolfgang vonBuddenbrock on his 80th birthday, March 25, 1964. It is based in part on more comprehensive reviews (Kinne 1963a, c, 1964a, b) dealing with the effects of temperature and salinity on marine and brackish-water animals.     

肉桂醛对增强辣椒疫霉病抗性的作用机制研究

该实验以辣椒疫霉菌(Phytophthora capsici)致病性菌株NJ01和‘苏椒5号’辣椒幼苗为研究对象,通过肉桂醛对辣椒疫霉菌的体外抑菌作用、室内侵染效果以及对辣椒幼苗防卫反应的调控作用,揭示肉桂醛对增强辣椒疫霉病抗性的作用机制。结果表明:(1)肉桂醛对实验所用致病性菌株NJ01的抑制中浓度(EC50)值为0.81mmol/L;肉桂醛处理可导致NJ01菌丝严重皱缩、畸形、断裂;PI染色显示NJ01菌丝出现明显的细胞死亡现象。(2)单独NJ01菌株接种的辣椒植株表现出明显病症(茎基部变黑褐色、干枯萎缩,植株倒伏,叶片脱落,生物量下降);而肉桂醛处理2h后接种NJ01菌株的辣椒植株长势良好,无明显病症,鲜重和叶绿素含量显著上升至对照水平。(3)与单独接种NJ01菌株处理相比,肉桂醛处理2h后接种NJ01菌株的辣椒植株体内抗氧化酶(CAT、SOD、POD)活性显著上升,抗氧化物质(GSH和ASA)含量显著增加。研究认为,肉桂醛可能通过抑制辣椒疫霉菌的生长及其对辣椒植株的侵染能力,同时调控辣椒植株防卫反应,进而提高辣椒对疫病抗性。     

Forming Patterns in Development without Morphogen Gradients: Scattered Differentiation and Sorting Out

Few mechanisms provide alternatives to morphogen gradients for producing spatial patterns of cells in development. One possibility is based on the sorting out of cells that initially differentiate in a salt and pepper mixture and then physically move to create coherent tissues. Here, we describe the evidence suggesting this is the major mode of patterning in Dictyostelium. In addition, we discuss whether convergent evolution could have produced a conceptually similar mechanism in other organisms.A limited number of processes are thought to regulate the differentiation of specialized cell types and their organization to form larger scale structures, such as organs or limbs, during embryonic development. First, early embryological experiments revealed a patterning process that depends on special “organizing” regions in the embryo. This idea was encapsulated as “positional information” and led to the concept of morphogen gradients (Fig. 1) (Wolpert 1996). In addition, cytoplasmic determinants have been shown to direct development along different lines when they are partitioned unequally between daughter cells by asymmetric cell division (Betschinger and Knoblich 2004). Finally, short-range inductive signaling can specify cells at a local level and when reiterated produces highly ordered structures (Simpson 1990; Freeman 1997; Meinhardt and Gierer 2000).Open in a separate windowFigure 1.Alternative ways of patterning cells during development. (A) Patterning by “positional information”: A group of undifferentiated cells is patterned by a morphogen diffusing from a pre-established source, producing a concentration gradient. Cells respond according to the local morphogen concentration, becoming red, white, or blue. (B, C) Patterning without positional information: This is a two-step process in which different cell types first differentiate mixed up with each other, and then sort out. The initial differentiation can be controlled by strictly local interactions between the cells, as in lateral inhibition (B), or by a global signal to which cells respond with different sensitivities and whose concentration they regulate by negative feedback (C). Once sorting has occurred, the global inducer forms a reverse gradient, which could then convey positional information for further patterning events.The question then arises of whether evolution has devised any further global patterning mechanisms. One possibility that has been repeatedly considered, but not firmly established as a general mechanism, is based on sorting out. In this process, pattern is produced in two steps: (1) Different cell types are initially specified from a precursor pool independent of their position to produce a salt and pepper mixture and (2) the mixture of cell types is resolved into discrete tissues by the physical movement and sorting out of the cells (Fig. 1). Consequently, this mechanism does not involve positional information. However, it can actually provide the conditions under which positional signaling and morphogen gradients can arise, if the resolved tissues then act as sources and sinks for signal molecules.We first describe the powerful evidence that this alternative patterning process is used during the developmental cycle of the social amoeba Dictyostelium discoideum, and then consider the possibility that this patterning strategy may be used more widely.     

Effects of transgenic expression of <Emphasis Type="Italic">Brevibacterium linens</Emphasis> methionine gamma lyase (MGL) on accumulation of <Emphasis Type="Italic">Tylenchulus semipenetrans and key aminoacid contents</Emphasis> in <Emphasis Type="Italic">Carrizo citrange</Emphasis>

Alpha-dioxygenases (α-DOX) catalyzing the primary oxygenation of fatty acids to oxylipins were recently found in plants. Here, the biological roles of the pepper α-DOX (Ca-DOX) gene, which is strongly induced during non-host pathogen infection in chili pepper, were examined. Virus-induced gene silencing demonstrated that down-regulation of Ca-DOX enhanced susceptibility to bacterial pathogens and suppressed the hypersensitive response via the suppression of pathogenesis-related genes such as PR4, proteinase inhibitor II and lipid transfer protein (PR14). Ca-DOX-silenced pepper plants also exhibited more retarded growth with lower epidermal cell numbers and reduced cell wall thickness than control plants. To better understand regulation of Ca-DOX, transgenic Arabidopsis plants harboring the β-glucuronidase (GUS) reporter gene driven from a putative Ca-DOX promoter were generated. GUS expression was significantly induced upon avirulent pathogen infection in transgenic Arabidopsis leaves, whereas GUS induction was relatively weak upon virulent pathogen treatment. After treatment with plant hormones, early and strong GUS expression was seen after treatment of salicylic acid, whereas ethylene and methyl jasmonate treatments produced relatively weak and late GUS signals. These results will enable us to further understand the role of α-DOX, which is important in lipid metabolism, defense responses, and growth development in plants.     

Phylogenetics and histology provide insight into damping-off infections of ‘Poblano’ pepper seedlings caused by <Emphasis Type="Italic">Fusarium</Emphasis> wilt in greenhouses

The ‘Poblano’ pepper crop is economically important in Mexico and throughout the world as it is used as a hot spice in food. The cultivated area of the ‘Poblano’ pepper crop is decreasing yearly for many reasons, among them a wilt disease commonly associated with Fusarium spp. This disease is a problem of field and greenhouse production plants. Moreover, it is not clear whether the pathogens that cause wilt in mature plants are the same as those involved in the damping-off symptoms and death of pepper seedlings in greenhouses. For this reason, the aim of the present study was to identify the causal agent of damping-off in pepper during seedling production, establish its relationship with the causal agent of wilting in mature plants, and determine whether histological damage in seedlings occurs. Isolates were recovered from the crown rot and stem base of 4-month-old infected ‘Poblano’ mature pepper plants and were identified using morphological and phylogenetic approaches. Fusarium oxysporum and F. solani were isolated from the crown rot and base stem, respectively. A pathogenicity test showed that both species caused damping-off in pepper seedlings. Histological studies with inoculated seedlings of both isolates showed several changes in the external cortex, epidermal cells, endodermis, Casparian strips, cell size, and xylem wall. Casparian strip rupture resulted in permeability loss and regulatory activity to maintain the cellular equilibrium inside the vascular bundles. Hence, according to these findings, producers should avoid seedling contamination by infected mature plants because the aggressiveness of Fusarium isolates can cause rapid seedling mortality.