A systems approach to morphogenesis in Arabidopsis thaliana: II. Modeling the regulation of shoot apical meristem structure

Two models of the mechanism maintaining a zonal structure in the shoot apical meristem (SAM) were built based on the analysis of experimental data on the interactions between CLV1, CLV2, CLV3, and WUS genes and the concepts of their role in this mechanism. The first model, a simple one-dimensional model with two morphogens, which is a variant of Wolpert’s French flag model [1], describes the regulation of zone distribution along the SAM vertical axis. Despite a number of simplifications, this model has stationary solutions with biologically meaningful interpretation. The simplifying assumptions were successively abandoned in constructing a two-dimensional model of the mechanism underlying the regulation of SAM structure. This model provides a better understanding of the distributed system that regulates the SAM structure, and allows more detailed formalization of the modern concepts and experimental data concerning this mechanism.

     

Dual CLAVATA3 peptides in Arabidopsis shoot stem cell signaling

Plant shoot stem cell pool is constantly maintained by a negative feedback loop through peptide-receptor mediated signaling pathway. CLAVATA3 (CLV3) encode a 96 aminoacid protein which is processed to 12-amino-acid or arabinosylated 13-amino-acid peptides, acting as a ligand signal to regulate stem cell homeostasis in the shoot apical meristem (SAM). Although arabinosylated 13-amino-acid CLV3 peptide (CLV3p) shows more significant binding affinity to its receptors and biological activities in the SAM, the physiological function of two mature forms of CLV3p remained an unresolved puzzle in the past decade due to the technical difficulties of arabinosylation modification in the peptide synthesis. Here, we analyzed the role of two mature CLV3 peptides with newly synthesized arabinosylated peptide. Beside shoot meristem phenotypes, arabinosylated CLV3p showed the conventional trait of CLV2-dependent root growth inhibition. Moreover, both 12-amino-acid and arabinosylated 13-amino-acid CLV3 peptides have analogous activities in shoot stem cell signaling. Notably, we demonstrated that non-arabinosylated 12-amino acid CLV3p can affect shoot stem cell signaling at the physiological level unlike previously suggested (Ohyama et al. 2009; Shinohara and Matsubayashi 2013; Shinohara and Matsubayashi 2015). Therefore, these results support the physiological role of the 12-amino-acid CLV3p in shoot stem cell signaling in the deficient condition of arabinosylated 13-amino-acid CLV3p in Arabidopsis thaliana.     

Improving methionine and ATP availability by <Emphasis Type="Italic">MET6</Emphasis> and <Emphasis Type="Italic">SAM2</Emphasis> co-expression combined with sodium citrate feeding enhanced SAM accumulation in <Emphasis Type="Italic">Saccharomyces cerevisiae</Emphasis>

S-adenosyl-l-methionine (SAM), biosynthesized from methionine and ATP, exhibited diverse pharmaceutical applications. To enhance SAM accumulation in S. cerevisiae CGMCC 2842 (wild type), improvement of methionine and ATP availability through MET6 and SAM2 co-expression combined with sodium citrate feeding was investigated here. Feeding 6 g/L methionine at 12 h into medium was found to increase SAM accumulation by 38 % in wild type strain. Based on this result, MET6, encoding methionine synthase, was overexpressed, which caused a 59 % increase of SAM. To redirect intracellular methionine into SAM, MET6 and SAM2 (encoding methionine adenosyltransferase) were co-expressed to obtain the recombinant strain YGSPM in which the SAM accumulation was 2.34-fold of wild type strain. The data obtained showed that co-expression of MET6 and SAM2 improved intracellular methionine availability and redirected the methionine to SAM biosynthesis. To elevate intracellular ATP levels, 6 g/L sodium citrate, used as an auxiliary energy substrate, was fed into the batch fermentation medium, and an additional 19 % increase of SAM was observed after sodium citrate addition. Meanwhile, it was found that addition of sodium citrate improved the isocitrate dehydrogenase activity which was associated with the intracellular ATP levels. The results demonstrated that addition of sodium citrate improved intracellular ATP levels which promoted conversion of methionine into SAM. This study presented a feasible approach with considerable potential for developing highly SAM-productive strains based on improving methionine and ATP availability.     

Over-expression of miR166a inhibits cotyledon formation in somatic embryos and promotes lateral root development in seedlings of Larix leptolepis

Somatic embryo (SE) regeneration is an ideal experimental system to realize rapid propagation of excellent clones and genetic improvement for perennial gymnosperms. In the present study, genes encoding the miRNA166 precursor were identified and LamiR166a was successfully transformed into the gymnosperm Larix leptolepis (L. leptolepis) and five LamiR166a over-expressed embryonic cell lines were screened out as stable embryo masses. As expected, the targets of miR166a, LaHDZ31-34, were all down-regulated in transgenic lines according to qRT-PCR results. The results showed that the percentage of normal SEs with 4–7 cotyledons was 77.0?% in wild type (WT) lines, but was reduced to 60.3?% in the pSuper::MIR166a lines with “cup-shaped” embryos comprised 7.0?% of WT and 20.7?% of transgenic embryos. Microscopic observation further showed that the intermediate region surrounded by the cotyledons was larger than in the control, with no upward bulge of the shoot apical meristem (SAM). The expression pattern of the two meristem marker genes CLAVATA (CLV) and WUSCHEL-related homeobox (WOX) were investigated. The results showed that the expression levels of WOX were three times higher in transgenic lines than in WT samples, which suggest that miR166a may indirectly regulate SAM development by directly affecting WOX expression. Besides, overexpression of LamiR166a clearly increased the rooting rate and promoted lateral root formation in L. leptolepis seedlings. These results may provide new insights into the regulatory role of miR166 in gymnosperms, and also new applications for forestry production in practice.     

Progress in the microbial production of <Emphasis Type="Italic">S</Emphasis>-adenosyl-<Emphasis Type="SmallCaps">l</Emphasis>-methionine

S-Adenosyl-l-methionine (SAM), which exists in all living organisms, serves as an activated group donor in a range of metabolic reactions, including trans-methylation, trans-sulfuration and trans-propylamine. Compared with its chemical synthesis and enzyme catalysis production, the microbial production of SAM is feasible for industrial applications. The current clinical demand for SAM is constantly increasing. Therefore, vast interest exists in engineering the SAM metabolism in cells for increasing product titers. Here, we provided an overview of updates on SAM microbial productivity improvements with an emphasis on various strategies that have been used to enhance SAM production based on increasing the precursor and co-factor availabilities in microbes. These strategies included the sections of SAM-producing microbes and their mutant screening, optimization of the fermentation process, and the metabolic engineering. The SAM-producing strains that were used extensively were Saccharomyces cerevisiae, Pichia pastoris, Candida utilis, Scheffersomyces stipitis, Kluyveromyces lactis, Kluyveromyces marxianus, Corynebacterium glutamicum, and Escherichia coli, in addition to others. The optimization of the fermentation process mainly focused on the enhancement of the methionine, ATP, and other co-factor levels through pulsed feeding as well as the optimization of nitrogen and carbon sources. Various metabolic engineering strategies using precise control of gene expression in engineered strains were also highlighted in the present review. In addition, some prospects on SAM microbial production were discussed.     

Relative Stabilities of C<Subscript>92</Subscript> IPR Fullerenes

The complete set of 86 isolated-pentagon-rule (IPR) isomers of C₉₂ has been described by the SAM1 quantum-chemical method, and their energetics checked by density functional theory at the B3LYP/6-31G* level. Although the lowest-energy cage is not identical in both approaches, it still exhibits D ₂ symmetry in both cases. As energetics themselves cannot produce reliable relative stabilities at high temperatures, entropy terms are also computed and the relative-stability problem is treated entirely in terms of the Gibbs function. The lowest-energy structure is not the most populated isomer at higher temperatures – it is replaced by a D ₃ structure. Further stability interchanges are possible at very high temperatures, when C ₃ and C ₁ structures are also important. There is a partial agreement of the computations with available observed data.     

Genome-wide identification and evolution of <Emphasis Type="Italic">TC1</Emphasis>/<Emphasis Type="Italic">Mariner</Emphasis> in the silkworm (<Emphasis Type="Italic">Bombyx mori</Emphasis>) genome

TC1/Mariner transposons belong to class II transposable elements (TEs) that use DNA-mediated “cut and paste” mechanism to transpose, and they have been identified in almost all organisms. Although silkworm (Bombyx mori) has a large amount of TC1/Mariner elements, the genome wide information of this superfamily in the silkworm is unknown. In this study, we have identified 2670 TC1/Mariner (Bmmar) elements in the silkworm genome. All the TEs were classified into 22 families by means of fgclust, a tool of repetitive sequence classification, seven of which was first reported in this study. Phylogenetic and structure analyses based on the catalytic domain (DDxD/E) of transposase sequences indicated that all members of TC1/Mariner were grouped into five subgroups: Mariner, Tc1, maT, DD40D and DD41D/E. Of these five subgroups, maT rather than Mariner possessed most members of TC1/Mariner (51.23%) in the silkworm genome. In particular, phylogenetic analysis and structure analysis revealed that Bmmar15 (DD40D) formed a new basal subgroup of TC1/Mariner element in insects, which was referred to as bmori. Furthermore, we concluded that DD40D appeared to intermediate between mariner and Tc1. Finally, we estimated the insertion time for each copy of TC1/Mariner in the silkworm and found that most of members were dramatically amplified during a period from 0 to 1 mya. Moreover, the detailed functional data analysis showed that Bmmar1, Bmmar6 and Bmmar9 had EST evidence and intact transposases. These implied that TC1/Mariner might have potential transpositional activity. In conclusion, this study provides some new insights into the landscape, origin and evolution of TC1/Mariner in the insect genomes.     

Clinical strains of <Emphasis Type="Italic">Lactobacillus</Emphasis> reduce the filamentation of <Emphasis Type="Italic">Candida albicans</Emphasis> and protect <Emphasis Type="Italic">Galleria mellonella</Emphasis> against experimental candidiasis

Candida albicans is the most common human fungal pathogen and can grow as yeast or filaments, depending on the environmental conditions. The filamentous form is of particular interest because it can play a direct role in adherence and pathogenicity. Therefore, the purpose of this study was to evaluate the effects of three clinical strains of Lactobacillus on C. albicans filamentation as well as their probiotic potential in pathogen-host interactions via an experimental candidiasis model study in Galleria mellonella. We used the reference strain Candida albicans ATCC 18804 and three clinical strains of Lactobacillus: L. rhamnosus strain 5.2, L. paracasei strain 20.3, and L. fermentum strain 20.4. First, the capacity of C. albicans to form hyphae was tested in vitro through association with the Lactobacillus strains. After that, we verified the ability of these strains to attenuate experimental candidiasis in a Galleria mellonella model through a survival curve assay. Regarding the filamentation assay, a significant reduction in hyphae formation of up to 57% was observed when C. albicans was incubated in the presence of the Lactobacillus strains, compared to a control group composed of only C. albicans. In addition, when the larvae were pretreated with Lactobacillus spp. prior to C. albicans infection, the survival rate of G. mellonela increased in all experimental groups. We concluded that Lactobacillus influences the growth and expression C. albicans virulence factors, which may interfere with the pathogenicity of these microorganisms.     

The expression level of anthocyanidin synthase determines the anthocyanin content of crabapple (<Emphasis Type="Italic">Malus</Emphasis> sp.) petals

In addition to contributing to the coloration of plant organs and their defense against herbivores, the consumption of anthocyanins in the human diet has a number of health benefits. Crabapple (Malus sp.) represents a valuable experimental model system to research the mechanisms and regulation of anthocyanin accumulation, in part due to the often vivid and varied petal and leaf coloration that is exhibited by various cultivars. The enzyme anthocyanidin synthase (ANS) plays a pivotal role in anthocyanin biosynthesis; however, the relationship between ANS expression and petal pigmentation has yet to be established in crabapple. To illuminate the mechanism of anthocyanin accumulation in crabapple petals, we evaluated the expression of two crabapple ANS allelic genes (McANS-1 and McANS-2) and the levels of anthocyanins in petals from cultivars with dark red (‘Royalty’) and white (‘Flame’) petals, as well as another (‘Radiant’) whose petals have an intermediate pink color. We determined that the expression of McANS in the three cultivars correlated with the variation of anthocyanin accumulation during different petal developmental stages. Furthermore, transgenic tobacco plants constitutively overexpressing one of the two McANS genes, McANS-1, had showed elevated anthocyanin accumulation and a deeper red coloration in their petals than those from untransformed control lines. In conclusion, we propose that McANS are responsible for anthocyanin accumulation during petal coloration in different crabapple cultivars.     

On the control mechanisms of the nitrite level in <Emphasis Type="Italic">Escherichia coli</Emphasis> cells: the mathematical model

Background

Due to a high toxicity of nitrite and its metabolites, it is of high interest to study mechanisms underlying the low NO₂ level maintenance in the cell. During anaerobic growth of Escherichia coli the main nitrite-reducing enzymes are NrfA and NirB nitrite reductases. NrfA reductase is localized in the cell periplasm and uses NO₂ as an electron acceptor to create a proton gradient; NirB reductase is restricted to the cytoplasm and metabolizes excessive nitrite inside the cell, the uptake of which is mediated by the transporter protein NirC. While it is known that these three systems, periplasmic, cytoplasmic and transport, determine nitrite uptake and assimilation in the cell as well as its excretion, little is known about their co-ordination.

Results

Using a mathematical model describing the nitrite utilization in E. coli cells cultured in a flow chemostat, the role of enzymes involved in nitrite metabolism and transport in controlling nitrite intracellular levels was investigated. It was demonstrated that the model adapted to the experimental data on expression of nrfA and nirB genes encoding NrfA and NirB nitrite reductases, can describe nitrite accumulation kinetics in the chemostat in the millimolar range of added substrate concentrations without any additional assumptions. According to the model, in this range, low intracellular nitrite level, weakly dependent on its concentration in the growth media, is maintained (mcM). It is not sufficient to consider molecular-genetic mechanisms of NrfA reductase activity regulation to describe the nitrite accumulation dynamics in the chemostat in the micromolar range (≤1 mM) of added nitrite concentrations. Analysis of different hypotheses has shown that the mechanism of local enzyme concentration change due to membrane potential-induced diffusion from the cytoplasm to the periplasm at low nitrite levels is sufficient to explain the nitrite accumulation dynamics in the chemostat.

Conclusions

At nitrite concentrations in the media more than 2 mM, the model adapted to the experimental data on nitrite utilization dynamics in E. coli cells cultured in the flow chemostat demonstrates the largest contribution of genetic mechanisms involved in nrf and nir operons activity regulation to the control of nitrite intracellular levels. The model predicts a significant contribution of the membrane potential to the periplasmic NrfA nitrite reductase activity regulation and nitrite utilization dynamics at substrate concentrations ≤1 mM.

     

Overexpression of Tomato Homolog of Glycolate/Glycerate Transporter Gene <Emphasis Type="Italic">PLGG1/AtLrgB</Emphasis> Leads to Reduced Chlorophyll Biosynthesis

LrgA and LrgB genes have been identified as new components in regulation of programmed cell death (PCD) in bacteria. While in Arabidopsis, it has been documented that AtLrgB plays a crucial role in chloroplast development and photorespiration by acting as a glycolate/glycerate translocator (PLGG1) in the chloroplast inner membrane. However, little is known about LrgB homologs in other plant species, especially those with fleshy fruits. In this study, a homologous gene of AtLrgB, here designated SlLrgB, was identified in tomato. Similar to AtLrgB, structure analysis suggests that the LrgA and LrgB genes have evolved into two domains of the SlLrgB protein. Expression pattern analysis showed that SlLrgB accumulated mainly in green tissues and could be regulated by light, hormone, and abiotic stress treatments. Compared to wild-type plants, parts of SlLrgB overexpression plants displayed etiolated leaves and a growth retardation phenotype, with significantly reduced chlorophyll content both in leaves and fruits. The qPCR results revealed that the SGR gene, which was associated with chlorophyll degradation, was severely repressed. Two key genes in the chlorophyll biosynthesis pathway, CAO and POR, were also suppressed in the SlLrgB overexpression plants. Taken together, we suggest that SlLrgB may play important roles in the regulation of chlorophyll metabolism pathways in tomato.     

Lipid metabolism in adipose tissue and liver from diet-induced obese rats: a comparison between <Emphasis Type="Italic">Wistar</Emphasis> and <Emphasis Type="Italic">Sprague-Dawley</Emphasis> strains

Some researchers have proposed important variations in adipose tissue among different strains of rats and mice in response to a high-caloric (hc) diet, but data concerning the mechanisms underlying these differences are scarce. The aim of the present research was to characterize different aspects of triacylglycerol (TG) metabolism and clock genes between Sprague-Dawley and Wistar rats. For this purpose, 16 male Sprague-Dawley and 16 male Wistar rats were divided into four experimental groups (n?=?8) and fed either a normal-caloric (nc) diet or a hc diet for 6 weeks. After sacrifice, liver and epididymal, perirenal, mesenteric, and subcutaneous adipose tissue depots were dissected, weighed and immediately frozen. Liver TG content was quantified, RNA extracted for gene expression analysis and fatty acid synthase enzyme activity measured. Two-way ANOVA and Student’s t test were used to perform the statistical analyses. Under hc feeding conditions, Wistar rats were more prone to fat accumulation in adipose tissue, especially in the epididymal fat depot, due to their increased lipogenesis and fatty acid uptake. By contrast, both strains of rats showed similarly fatty livers after hc feeding. Peripheral clock machinery seems to be a potential explanatory mechanism for Wistar and Sprague-Dawley strain differences. In conclusion, Wistar strain seems to be the best choice as animal model in dietary-induced obesity studies.     

Genome-wide identification and expression analysis of the GRAS family proteins in <Emphasis Type="Italic">Medicago truncatula</Emphasis>

The GRAS gene family performs a variety of functions in plant growth and development processes, and they also play essential roles in plant response to environmental stresses. Medicago truncatula is a diploid plant with a small genome used as a model organism. Despite the vital role of GRAS genes in plant growth regulation, few studies on these genes in M. truncatula have been conducted to date. Using the M. truncatula reference genome data, we identified 68 MtGRAS genes, which were classified into 16 groups by phylogenetic analysis, located on eight chromosomes. The structure analysis indicated that MtGRAS genes retained a relatively constant exon–intron composition during the evolution of the M. truncatula genome. Most of the closely related members in the phylogenetic tree had similar motif compositions. Different motifs distributed in different groups of the MtGRAS genes were the sources of their functional divergence. Twenty-eight MtGRAS genes were expressed in six tissues, namely root, bud, blade, seedpod, nodule, and flower tissues, suggesting their putative function in many aspects of plant growth and development. Nine MtGRAS genes were upregulated under cold, freezing, drought, ABA, and salt stress treatments, indicating that they play vital roles in the response to abiotic stress in M. truncatula. Our study provides valuable information that can be utilized to improve the quality and agronomic benefits of M. truncatula and other plants.     

Enhancers in the <Emphasis Type="Italic">Peril</Emphasis> lincRNA locus regulate distant but not local genes

Background

Recently, it has become clear that some promoters function as long-range regulators of gene expression. However, direct and quantitative assessment of enhancer activity at long intergenic noncoding RNA (lincRNA) or mRNA gene bodies has not been performed. To unbiasedly assess the enhancer capacity across lincRNA and mRNA loci, we performed a massively parallel reporter assay (MPRA) on six lincRNA loci and their closest protein-coding neighbors.

Results

For both gene classes, we find significantly more MPRA activity in promoter regions than in gene bodies. However, three lincRNA loci, Lincp21, LincEnc1, and Peril, and one mRNA locus, Morc2a, display significant enhancer activity within their gene bodies. We hypothesize that such peaks may mark long-range enhancers, and test this in vivo using RNA sequencing from a knockout mouse model and high-throughput chromosome conformation capture (Hi-C). We find that ablation of a high-activity MPRA peak in the Peril gene body leads to consistent dysregulation of Mccc1 and Exosc9 in the neighboring topologically associated domain (TAD). This occurs irrespective of Peril lincRNA expression, demonstrating this regulation is DNA-dependent. Hi-C confirms long-range contacts with the neighboring TAD, and these interactions are altered upon Peril knockout. Surprisingly, we do not observe consistent regulation of genes within the local TAD. Together, these data suggest a long-range enhancer-like function for the Peril gene body.

Conclusions

A multi-faceted approach combining high-throughput enhancer discovery with genetic models can connect enhancers to their gene targets and provides evidence of inter-TAD gene regulation.

     

Up-Regulation of Antimicrobial Peptides Gallerimycin and Galiomicin in <Emphasis Type="Italic">Galleria mellonella</Emphasis> Infected with <Emphasis Type="Italic">Candida</Emphasis> Yeasts Displaying Different Virulence Traits

Galleria mellonella has been described as a cheap and an easy-to-reproduce model for the study of fungal infections. We hypothesized that yeasts with higher virulence potential decrease survival and significantly trigger an immune response in G. mellonella through the regulation of innate immunity-related genes encoding antimicrobial peptides (AMPs) such as gallerimycin and galiomicin. Candida albicans SC5314 and Candida dubliniensis CBS 7987, selected because of their different virulence potential, were used for a killing assay followed by the determination of gene expression using qPCR. In vivo results confirmed a significantly (p?=?0.0321) lower pathogenicity for C. dubliniensis than for C. albicans. Accordingly, the induction of C. dubliniensis AMPs was lower at all the selected time points post-infection (1 h, 24 h, 48 h). Moreover, we observed an extremely high regulation of the galiomicin gene compared to the gallerimycin one, suggesting a different role of the tested AMPs in protecting G. mellonella from candidiasis.