The Cytokinins BAP and 2-iP Modulate Different Molecular Mechanisms on Shoot Proliferation and Root Development in Lemongrass (Cymbopogon citratus).

The known activities of cytokinins (CKs) are promoting shoot multiplication, root growth inhibition, and delaying senescence. 6-Benzylaminopurine (BAP) has been the most effective CK to induce shoot proliferation in cereal and grasses. Previously, we reported that in lemongrass (Cymbopogon citratus) micropropagation, BAP 10 µM induces high shoot proliferation, while the natural CK 6-(γ,γ-Dimethylallylamino)purine (2-iP) 10 µM shows less pronounced effects and developed rooting. To understand the molecular mechanisms involved, we perform a protein-protein interaction (PPI) network based on the genes of Brachypodium distachyon involved in shoot proliferation/repression, cell cycle, stem cell maintenance, auxin response factors, and CK signaling to analyze the molecular mechanisms in BAP versus 2-iP plants. A different pattern of gene expression was observed between BAP- versus 2-iP-treated plants. In shoots derived from BAP, we found upregulated genes that have already been demonstrated to be involved in de novo shoot proliferation development in several plant species; CK receptors (AHK3, ARR1), stem cell maintenance (STM, REV and CLV3), cell cycle regulation (CDKA-CYCD3 complex), as well as the auxin response factor (ARF5) and CK metabolism (CKX1). In contrast, in the 2-iP culture medium, there was an upregulation of genes involved in shoot repression (BRC1, MAX3), ARR4, a type A-response regulator (RR), and auxin metabolism (SHY2).

Sex determination in papaya: Current status and perspectives.

Papaya (Carica papaya L.) is an economically significant plant that produces fruit consumed worldwide due to its organoleptic characteristics. Since their commercial production, papaya fruits have faced several problems, such as pests, which have been partly resolved using transgenic varieties. Nevertheless, a principal challenge in this cultivation is the plant's sex determination. The sex issue in papaya is complex because papaya flowers can bear three sex forms: male, female, and hermaphrodite, which affects their fruit production, shape, and yield. Fruits from hermaphrodite plants are preferred more by consumers than female ones, and male plants rarely produce fruits without commercial value. Chromosomes are responsible for sex determination in papaya, denoted as XY for male, XX for female, and XYh for hermaphrodite. However, genes related to sex have been reported but are not conclusive. Factors such as the environment, hormones, and genetic and epigenetic background can also affect sex expression. Therefore, in this review, we will discuss recent research on the sex of papaya, from reported genes to date, their biology, and sexing approaches using molecular markers and their advantages.

Basics on network theory to analyze biological systems: a hands-on outlook.

Biological processes result from interactions among molecules and cell-to-cell communications. In the last 50 years, network theory has empowered advances in understanding molecular networks' structure and dynamics that regulate biological systems. Adopting a network data analysis point of view at more laboratories might enrich their research capacity to generate forward working hypotheses. This work briefly describes network theory origins and provides basic graph analysis principles in biological systems, specific centrality measurements, and the main models for network structures. Also, we describe a workflow employing user-friendly free platforms to process, construct, and analyze transcriptome data from a network perspective. With this assay, we expect to encourage the implementation of network theory analysis on biological data in everyday laboratory research.

Review: Isoprenoid and aromatic cytokinins in shoot branching.

Shoot branching is an important event of plant development that defines growth and reproduction. The BRANCHED1 gene (BRC1/TB1/FC1) is crucial for this process. Within the phytohormones, cytokinins directly activate axillary buds to promote shoot branching. In addition, strigolactones and auxins inhibit bud outgrowth. This review addresses the involvement of aromatic and isoprenoid cytokinins in shoot branching. And how auxins and strigolactones contribute to regulating this process also. The results obtained by others and our working group with lemongrass (Cymbopogon citratus) show that cytokinins affect both shoot and root apical meristem development, consistent with other plant species. However, many questions remain about how cytokinins and strigolactones antagonistically regulate BRC1 gene expression. Additionally, many details of the interaction among cytokinins, auxins, and strigolactones need to be clarified. We will gain a more comprehensive scheme of bud outgrowth with these details.

Pas de Trois: An Overview of Penta-, Tetra-, and Octo-Tricopeptide Repeat Proteins From Chlamydomonas reinhardtii and Their Role in Chloroplast Gene Expression.

Penta-, Tetra-, and Octo-tricopeptide repeat (PPR, TPR, and OPR) proteins are nucleus-encoded proteins composed of tandem repeats of 35, 34, and 38-40 amino acids, respectively. They form helix-turn-helix structures that interact with mRNA or other proteins and participate in RNA stabilization, processing, maturation, and act as translation enhancers of chloroplast and mitochondrial mRNAs. These helical repeat proteins are unevenly present in plants and algae. While PPR proteins are more abundant in plants than in algae, OPR proteins are more abundant in algae. In Arabidopsis, maize, and rice there have been 450, 661, and 477 PPR proteins identified, respectively, which contrasts with only 14 PPR proteins identified in Chlamydomonas reinhardtii. Likewise, more than 120 OPR proteins members have been predicted from the nuclear genome of C. reinhardtii and only one has been identified in Arabidopsis thaliana. Due to their abundance in land plants, PPR proteins have been largely characterized making it possible to elucidate their RNA-binding code. This has even allowed researchers to generate engineered PPR proteins with defined affinity to a particular target, which has served as the basis to develop tools for gene expression in biotechnological applications. However, fine elucidation of the helical repeat proteins code in Chlamydomonas is a pending task. In this review, we summarize the current knowledge on the role PPR, TPR, and OPR proteins play in chloroplast gene expression in the green algae C. reinhardtii, pointing to relevant similarities and differences with their counterparts in plants. We also recapitulate on how these proteins have been engineered and shown to serve as mRNA regulatory factors for biotechnological applications in plants and how this could be used as a starting point for applications in algae.

RNA polymerases in strict endosymbiont bacteria with extreme genome reduction show distinct erosions that might result in limited and differential promoter recognition.

Strict endosymbiont bacteria present high degree genome reduction, retain smaller proteins, and in some instances, lack complete functional domains compared to free-living counterparts. Until now, the mechanisms underlying these genetic reductions are not well understood. In this study, the conservation of RNA polymerases, the essential machinery for gene expression, is analyzed in endosymbiont bacteria with extreme genome reductions. We analyzed the RNA polymerase subunits to identify and define domains, subdomains, and specific amino acids involved in precise biological functions known in Escherichia coli. We also perform phylogenetic analysis and three-dimensional models over four lineages of endosymbiotic proteobacteria with the smallest genomes known to date: Candidatus Hodgkinia cicadicola, Candidatus Tremblaya phenacola, Candidatus Tremblaya Princeps, Candidatus Nasuia deltocephalinicola, and Candidatus Carsonella ruddii. We found that some Hodgkinia strains do not encode for the RNA polymerase α subunit. The rest encode genes for α, ß, ß', and σ subunits to form the RNA polymerase. However, 16% shorter, on average, respect their orthologous in E. coli. In the α subunit, the amino-terminal domain is the most conserved. Regarding the ß and ß' subunits, both the catalytic core and the assembly domains are the most conserved. However, they showed compensatory amino acid substitutions to adapt to changes in the σ subunit. Precisely, the most erosive diversity occurs within the σ subunit. We identified broad amino acid substitution even in those recognizing and binding to the -10-box promoter element. In an overall conceptual image, the RNA polymerase from Candidatus Nasuia conserved the highest similarity with Escherichia coli RNA polymerase and their σ70. It might be recognizing the two main promoter elements (-10 and -35) and the two promoter accessory elements (-10 extended and UP-element). In Candidatus Carsonella, the RNA polymerase could recognize all the promoter elements except the -10-box extended. In Candidatus Tremblaya and Hodgkinia, due to the α carboxyl-terminal domain absence, they might not recognize the UP-promoter element. We also identified the lack of the ß flap-tip helix domain in most Hodgkinia's that suggests the inability to bind the -35-box promoter element.

Limited oxygen conditions as an approach to scale-up and improve D and L-lactic acid production in mineral media and avocado seed hydrolysates with metabolically engineered Escherichia coli.

The effectiveness of micro-aeration on lactate (LA) production by metabolically engineered Escherichia coli was evaluated in 1 L bioreactors containing mineral media and glucose (70 g/L). Volumetric oxygen transfer coefficients (kLa) between 12.6 and 28.7 h-1 increased the specific growth rate (µ) and volumetric productivity (QLA) by 300 and 400%, respectively, without a significant decrease in lactate yield (YLA), when compared with non-aerated fermentations. A kLa of 12.6 h-1 was successfully used as a criterion to scale-up the production of L and D-lactate from 1 to 11 and 130 L. Approximately constant QLA and YLA values were obtained throughout the fermentation scale-up process. Furthermore, a D-lactogenic fermentation was carried out in 1 L bioreactors using avocado seed hydrolysate as a culture medium under the same kLa value, displaying high QLA and YLA.

RegulomePA: a database of transcriptional regulatory interactions in Pseudomonas aeruginosa PAO1.

We present RegulomePA, a database that contains biological information on regulatory interactions between transcription factors (TFs), sigma factor (SFs) and target genes in Pseudomonas aeruginosa PAO1. RegulomePA consists of 4827 regulatory interactions between 2831 nodes, which represent the interactions of TFs and SFs with their target genes, from the total of predicted RegulomePA including 27.27% of the TFs, 54.16% of SFs and 50.8% of the total genes. Each entry in the database corresponds to one node in the network and provides comprehensive details about the gene and its regulatory interactions such as gene description, nucleotide sequence, genome-strand position and links to other databases as well as the type of regulation it exerts or to which it is being subject (repression or activation), the associated experimental evidence and references, and topological information. Additionally, RegulomePA provides a way to recover information on the regulatory circuits of the network to which a gene pertains and also makes available the source codes to analyze the topology of any other regulatory network. The database will be updated yearly, by our team, with the contributions from ourselves and users, since the users are provided with an interactive platform where they can add interactions to the regulatory network feeding it with their respective references. Database URL: www.regulome.pcyt.unam.mx.

Quantitative modeling of the interplay between synthetic gene circuits and host physiology: experiments, results, and prospects.

A primary goal of synthetic biology is to develop gene circuits that perform their intended functions despite variations in the growth conditions. However, this has turned out to be more complicated than it originally seemed because there is a complex interplay between the operation of synthetic gene circuits and the global physiology of host cells. Mathematical models provide an avenue to disentangle the intricacies of this phenomenon and guide the design of synthetic circuits that robustly perform in a variety of conditions. In this work, we review quantitative modeling approaches that have been used to rationalize and predict experimental observations resulting from circuit-to-circuit and circuit-host interactions in bacteria.

Modeling Asymmetric Cell Division in Caulobacter crescentus Using a Boolean Logic Approach.

Caulobacter crescentus is a model organism for the study of asymmetric division and cell type differentiation, as its cell division cycle generates a pair of daughter cells that differ from one another in their morphology and behavior. One of these cells (called stalked) develops a structure that allows it to attach to solid surfaces and is the only one capable of dividing, while the other (called swarmer) develops a flagellum that allows it to move in liquid media and divides only after differentiating into a stalked cell type. Although many genes, proteins, and other molecules involved in the asymmetric division exhibited by C. crescentus have been discovered and characterized for several decades, it remains as a challenging task to understand how cell properties arise from the high number of interactions between these molecular components. This chapter describes a modeling approach based on the Boolean logic framework that provides a means for the integration of knowledge and study of the emergence of asymmetric division. The text illustrates how the simulation of simple logic models gives valuable insight into the dynamic behavior of the regulatory and signaling networks driving the emergence of the phenotypes exhibited by C. crescentus. These models provide useful tools for the characterization and analysis of other complex biological networks.

Consensus architecture of promoters and transcription units in Escherichia coli: design principles for synthetic biology.

Genetic information in genomes is ordered, arranged in such a way that it constitutes a code, the so-called cis regulatory code. The regulatory machinery of the cell, termed trans-factors, decodes and expresses this information. In this way, genomes maintain a potential repertoire of genetic programs, parts of which are executed depending on the presence of active regulators in each condition. These genetic programs, executed by the regulatory machinery, have functional units in the genome delimited by punctuation-like marks. In genetic terms, these informational phrases correspond to transcription units, which are the minimal genetic information expressed consistently from initiation to termination marks. Between the start and final punctuation marks, additional marks are present that are read by the transcriptional and translational machineries. In this work, we look at all the experimentally described and predicted genetic elements in the bacterium Escherichia coli K-12 MG1655 and define a comprehensive architectural organization of transcription units to reveal the natural genome-design and to guide the construction of synthetic genetic programs.

Transcription Factors Exhibit Differential Conservation in Bacteria with Reduced Genomes.

The description of transcriptional regulatory networks has been pivotal in the understanding of operating principles under which organisms respond and adapt to varying conditions. While the study of the topology and dynamics of these networks has been the subject of considerable work, the investigation of the evolution of their topology, as a result of the adaptation of organisms to different environmental conditions, has received little attention. In this work, we study the evolution of transcriptional regulatory networks in bacteria from a genome reduction perspective, which manifests itself as the loss of genes at different degrees. We used the transcriptional regulatory network of Escherichia coli as a reference to compare 113 smaller, phylogenetically-related γ-proteobacteria, including 19 genomes of symbionts. We found that the type of regulatory action exerted by transcription factors, as genomes get progressively smaller, correlates well with their degree of conservation, with dual regulators being more conserved than repressors and activators in conditions of extreme reduction. In addition, we found that the preponderant conservation of dual regulators might be due to their role as both global regulators and nucleoid-associated proteins. We summarize our results in a conceptual model of how each TF type is gradually lost as genomes become smaller and give a rationale for the order in which this phenomenon occurs.

Some physicochemical and rheological properties of starch isolated from avocado seeds.

Seeds from avocado (Persea americana Miller) fruit are a waste byproduct of fruit processing. Starch from avocado seed is a potential alternative starch source. Two different extraction solvents were used to isolate starch from avocado seeds, functional and rheological characteristics measured for these starches, and comparisons made to maize starch. Avocado seed powder was suspended in a solution containing 2 mM Tris, 7.5 mM NaCl and 80 mM NaHSO3 (solvent A) or sodium bisulphite solution (1500 ppm SO2, solvent B). Solvent type had no influence (p>0.05) on starch properties. Amylose content was 15-16%. Gelatinization temperature range was 56-74 °C, peak temperature was 65.7 °C, and transition enthalpy was 11.4-11.6J/g. At 90 °C, solubility was 19-20%, swelling power 28-30 g water/g starch, and water absorption capacity was 22-24 g water/g starch. Pasting properties were initial temperature 72 °C; maximum viscosity 380-390 BU; breakdown -2 BU; consistency 200 BU; and setback 198 BU. Avocado seed starch dispersions (5% w/v) were characterized as viscoelastic systems, with G'>G″. Avocado seed starch has potential applications as a thickening and gelling agent in food systems, as a vehicle in pharmaceutical systems and an ingredient in biodegradable polymers for food packaging.

Dynamical modeling of the cell cycle and cell fate emergence in Caulobacter crescentus.

The division of Caulobacter crescentus, a model organism for studying cell cycle and differentiation in bacteria, generates two cell types: swarmer and stalked. To complete its cycle, C. crescentus must first differentiate from the swarmer to the stalked phenotype. An important regulator involved in this process is CtrA, which operates in a gene regulatory network and coordinates many of the interactions associated to the generation of cellular asymmetry. Gaining insight into how such a differentiation phenomenon arises and how network components interact to bring about cellular behavior and function demands mathematical models and simulations. In this work, we present a dynamical model based on a generalization of the Boolean abstraction of gene expression for a minimal network controlling the cell cycle and asymmetric cell division in C. crescentus. This network was constructed from data obtained from an exhaustive search in the literature. The results of the simulations based on our model show a cyclic attractor whose configurations can be made to correspond with the current knowledge of the activity of the regulators participating in the gene network during the cell cycle. Additionally, we found two point attractors that can be interpreted in terms of the network configurations directing the two cell types. The entire network is shown to be operating close to the critical regime, which means that it is robust enough to perturbations on dynamics of the network, but adaptable to environmental changes.

Engineering Escherichia coli K12 MG1655 to use starch.

BACKGROUND: To attain a sustainable bioeconomy, fuel, or valuable product, production must use biomass as substrate. Starch is one of the most abundant biomass resources and is present as waste or as a food and agroindustry by-product. Unfortunately, Escherichia coli, one of the most widely used microorganisms in biotechnological processes, cannot use starch as a carbon source. RESULTS: We engineered an E. coli strain capable of using starch as a substrate. The genetic design employed the native capability of the bacterium to use maltodextrins as a carbon source plus expression and secretion of its endogenous α-amylase, AmyA, in an adapted background. Biomass production improved using 35% dissolved oxygen and pH 7.2 in a controlled bioreactor. CONCLUSION: The engineered E. coli strain can use starch from the milieu and open the possibility of optimize the process to use agroindustrial wastes to produce biofuels and other valuable chemicals.

Structural comparison of biological networks based on dominant vertices.

It is a current practice to organize biological data in a network structure where vertices represent biological components and arrows represent their interactions. A great diversity of graph theoretical notions, such as clustering coefficient, network motifs, centrality, degree distribution, etc., have been developed in order to characterize the structure of these networks. However, none of the existent characterizations allow us to determine global similarity among networks of different sizes. It is the aim of the present paper to introduce a mathematical tool to compare networks not only with regard to their topological structure, but also in their dynamical capabilities. For this reason we aim to propose a pseudo-distance between networks, built around the notions of determination and dominancy, concepts recently introduced in the context of regulatory dynamics on networks. We use our proposed pseudo-distance to compare networks from the following bacteria: E. coli, B. subtilis, P. aeruginosa, M. tuberculosis, S. aureus and C. glutamicum. We also use this pseudo-distance to compare these real bacterial networks with equivalent homogeneous, scale-free and geometric three dimensional random networks. We found that even when bacterial networks are characterized with different levels of detail, have different sizes and represent different aspects of the organisms, the proposed pseudo-distance captures all these characteristics, and indicates how similar they are or not from random networks.

Hierarchical dynamics of a transcription factors network in E. coli.

In the present work we study the transcription-factor regulatory network that controls the synthesis of flagella in E. coli. Our objective is to address how the transcription-factor dynamics (in terms of their promoter activities and associated rates) correlate with their positions in the hierarchical organization of this regulatory network. Our results suggest that global-regulator promoters express at higher rates than those of local regulators, particularly when the bacterial populations are actively growing. Furthermore, promoter activity decreases together with the rate of cellular division. And finally, local-regulator promoters reach their maximal activity later than global-regulator promoters do. In summary, our results suggest a strong correlation between promoter activities and their hierarchical organization in this particular regulatory network.

Regulatory design governing progression of population growth phases in bacteria.

It has long been noted that batch cultures inoculated with resting bacteria exhibit a progression of growth phases traditionally labeled lag, exponential, pre-stationary and stationary. However, a detailed molecular description of the mechanisms controlling the transitions between these phases is lacking. A core circuit, formed by a subset of regulatory interactions involving five global transcription factors (FIS, HNS, IHF, RpoS and GadX), has been identified by correlating information from the well- established transcriptional regulatory network of Escherichia coli and genome-wide expression data from cultures in these different growth phases. We propose a functional role for this circuit in controlling progression through these phases. Two alternative hypotheses for controlling the transition between the growth phases are first, a continuous graded adjustment to changing environmental conditions, and second, a discontinuous hysteretic switch at critical thresholds between growth phases. We formulate a simple mathematical model of the core circuit, consisting of differential equations based on the power-law formalism, and show by mathematical and computer-assisted analysis that there are critical conditions among the parameters of the model that can lead to hysteretic switch behavior, which--if validated experimentally--would suggest that the transitions between different growth phases might be analogous to cellular differentiation. Based on these provocative results, we propose experiments to test the alternative hypotheses.

The Regulatory Network of Pseudomonas aeruginosa.

BACKGROUND: Pseudomonas aeruginosa is an important bacterial model due to its metabolic and pathogenic abilities, which allow it to interact and colonize a wide range of hosts, including plants and animals. In this work we compile and analyze the structure and organization of an experimentally supported regulatory network in this bacterium. RESULTS: The regulatory network consists of 690 genes and 1020 regulatory interactions between their products (12% of total genes: 54% sigma and 16% of transcription factors). This complex interplay makes the third largest regulatory network of those reported in bacteria. The entire network is enriched for activating interactions and, peculiarly, self-activation seems to occur more prominent for transcription factors (TFs), which contrasts with other biological networks where self-repression is dominant. The network contains a giant component of 650 genes organized into 11 hierarchies, encompassing important biological processes, such as, biofilms formation, production of exopolysaccharide alginate and several virulence factors, and of the so-called quorum sensing regulons. CONCLUSIONS: The study of gene regulation in P. aeruginosa is biased towards pathogenesis and virulence processes, all of which are interconnected. The network shows power-law distribution -input degree -, and we identified the top ten global regulators, six two-element cycles, the longest paths have ten steps, six biological modules and the main motifs containing three and four elements. We think this work can provide insights for the design of further studies to cover the many gaps in knowledge of this important bacterial model, and for the design of systems strategies to combat this bacterium.

Regulatory dynamics of standard two-component systems in bacteria.

Complex cellular networks regulate metabolism, environmental adaptation, and phenotypic changes in biological systems. Among the elements forming regulatory networks in bacteria are regulatory proteins such as transcription factors, which respond to exogenous and endogenous conditions. To perceive their surroundings, bacteria have evolved sensory regulatory systems of two-components. The archetype of these systems is made up of two proteins--a signal sensor and a response regulator-whose genes are usually located together in a single transcription unit. These units switch transcriptional programs in response to environmental conditions. Here, we study 14 two-component systems in Escherichia coli, which have been experimentally characterized with respect to their transcriptional regulation and their perceived signal. Given that the activity of these sensory units is connected to the rest of the transcriptional network, we first classify them as autonomous, semiautonomous or dependent, according to whether or not they use additional regulators to be transcribed. Next, we use discrete-time models to simulate their qualitative regulatory dynamics in response to their transcriptional regulation and to the activation of these systems by their cognate signals. Compared to more traditional ordinary differential equations method, ours has the advantage of being computationally simple and mathematically tractable, while keeping the ability to reproduce the phenomenology described by non-linear models. The aim of the present work is not the study of all possible behaviors of these two-component systems, but to exemplify those behaviors reported in the literature. On the other hand, most of these systems are auto-activating switches, a property that distinguishes them from the other transcription factors in the regulatory network, which are mostly auto-repressing. Based on the data, our models show dynamic behaviors that explain how most of these sensory systems convey abilities for multistationarity, and these dynamic properties could explain the phenotypic heterogeneity observed in bacterial populations. Our results are likely to have an impact in the design of synthetic signaling modules.