Identification and functional analysis of anthocyanin biosynthesis genes in <Emphasis Type="Italic">Phalaenopsis</Emphasis> hybrids

Phalaenopsis species are among the most popular potted flowers for their fascinating flowers. When their whole-genome sequencing was completed, they have become useful for studying the molecular mechanism of anthocyanin biosynthesis. Here, we identified 49 candidate anthocyanin synthetic genes in the Phalaenopsis genome. Our results showed that duplication events might contribute to the expansion of some gene families, such as the genes encoding chalcone synthase (PeCHS), flavonoid 3′-hydroxylase (PeF3′H), and myeloblastosis (PeMYB). To elucidate their functions in anthocyanin biosynthesis, we conducted a global expression analysis. We found that anthocyanin synthesis occurred during the very early flower development stage and that the flavanone 3-hydroxylase (F3H), F3′H, and dihydroflavonol 4-reductase (DFR) genes played key roles in this process. Over-expression of Phalaenopsis flavonoid 3′,5′-hydroxylase (F3′5′H) in petunia showed that it had no function in anthocyanin production. Furthermore, global analysis of sequences and expression patterns show that the regulatory genes are relatively conserved and might be important in regulating anthocyanin synthesis through different combined expression patterns. To determine the functions of MYB2, 11, and 12, we over-expressed them in petunia and performed yeast two-hybrid analysis with anthocyanin (AN)1 and AN11. The MYB2 protein had strong activity in regulating anthocyanin biosynthesis and induced significant pigment accumulation in transgenic plant petals, whereas MYB11 and MYB12 had lower activities. Our work provided important improvement in the understanding of anthocyanin biosynthesis and established a foundation for floral colour breeding in Phalaenopsis through genetic engineering.     

Enzymatic depolymerization of <Emphasis Type="Italic">N</Emphasis>-succinylchitosan

A complex of chitinolytic enzymes of Streptomyces kurssanovii and also lysozyme and Celloviridin, an industrial cellulase preparation, were demonstrated to provide for an enzymatic hydrolysis of N-succinylchitosan. Our studies were carried out on a high-molecular N-succinylchitosan with M of 390 kDa and a substitution degree of 0.8 as a substrate. All the enzymatic preparations were shown to be suitable for the obtaining of low-molecular derivatives of N-succinylchitosan. The complex of enzymes from S. kurssanovii showed the greatest activity: they reduced the characteristic viscosity of initial solution of the substrate by 78% for 30 min. A biodegradation of N-succinylchitosan of various molecular masses was shown to proceed under the action of lysozyme, and the cleavage reaction was revealed to decelerate at a decrease of the polymer molecular mass. A use of N-succinylchitosan in a complex with drugs for a prolongation of their action in a live organism was presumed.     

Neuro-and psychotropic activity of <Emphasis Type="Italic">N</Emphasis>-uronoylamino acids and <Emphasis Type="Italic">N</Emphasis>-uronoylpeptides

Peculiarities of the rat behavior were studied in a series of experimental stress models after a systemic administration of new N-uronoyl derivatives of amino acids. The psychotropic effect was shown to be determined by the nature of the amino acid fragment. N-(1,2:3,4-Di-O-isopropylidene-α-D-galactopyraneuronoyl)-glycylglycine exhibited an anxiolytic effect more pronounced than that of pyracetam, whereas N-(1,2:3,4-di-O-isopropilidene-α-D-galactopyranuronoyl)-glycylglutamic acid has antidepressant action stronger than that of amitriptyline. Mechanisms for the psychotropic effects of the examined derivatives are discussed.     

The lignin synthesis related genes and lodging resistance of <Emphasis Type="Italic">Fagopyrum esculentum</Emphasis>

Lignin is closely related to the lodging resistance of common buckwheat (Fagopyrum esculentum Moench.). However, the characteristics of lignin synthesis related genes have not yet been reported. We investigated the lignin biosynthesis gene expression, activities of related enzymes, and accumulation of lignin monomers during branching stage, bloom stage, and milky ripe stage by real-time quantitative PCR, UVspectrophotometry, and gas chromatography-mass spectrometry in the 2^nd internode of three common buckwheat cultivars with different lodging resistance. The results showed that lignin content and the activity of phenylalanine ammonia lyase (PAL), 4-coumarate: CoA ligase (4CL), cinnamyl alcohol dehydrogenase (CAD) and peroxidase (POD) were closely related to the lodging resistance of common buckwheat. Further, we studied gene expression of cinnamate 4-hydroxylase (C4H), caffeoyl-CoA O-methyltransferase (CCoAOMT), ferulate 5-hydroxylase (F5H), cinnamoyl-CoA reductase (CCR), and caffeic acid O-methyltransferase (COMT). The lignin biosynthesis genes were divided into three classes according to their expression pattern: 1) expression firstly increasing and then descending (PAL, 4CL, CAD, C4H, CCoAOMT, F5H, and CCR), 2) expression remaining constant during maturation (C3H), and 3) expression decreasing with maturation (COMT). The present study provides preliminary insights into the expression of lignin biosynthesis genes in common buckwheat, laying a foundation for further understanding the lignin biosynthesis.     

Influence of genetic variants modulating dopamine activity on the brain processing of auditory information (the <Emphasis Type="Italic">P</Emphasis>300 paradigm)

We studied the influence of catechol-O-methyl transferase gene (COMT) polymorphism on the parameters of components of auditory evoked potentials (EPs) recorded under the conditions of selective attention in mentally healthy relatives of patients with endogenous psychosis. A smaller N100 latency of the EP in response to an insignificant stimulus, smaller N100 amplitude of the EP in response to a significant stimulus, smaller N200 latency, and larger P300 amplitude were found in subjects with the Met/Met genotype (with a higher dopamine activity), and a larger P200 latency was found in Val/Val genotype carriers. There was no influence of MMN gene polymorphism on the mismatch negativity.     

Development of schemes of induced mutagenesis for improving the productivity of <Emphasis Type="Italic">Aspergillus</Emphasis> strains producing amylolytic enzymes

In order to improve the biosynthesis of amylolytic enzymes by industrial Aspergillus strains, the efficiency of stepwise application of the following methods of induced mutagenesis was studied: ultraviolet (UV) irradiation, gamma irradiation, and treatment with N-methyl-N-nitro-N-nitrosoguanidine (NG). It was found that at the early stages of mutagenesis of the glucoamylase-producing A. awamori strain UV and NG, used either alone or in combination, were efficient enough as mutagenic agents, providing an increase of glucoamylase activity by 30–35 and 50–60%, respectively. At the later stages, serial UV mutagenesis and gamma-irradiation showed high efficiency for both A. awamori, and A. oryzae strains. Gamma-mutagenesis of Aspergillus strains using a cobalt source provided the most stable and highly active strains retaining 90–95% of their activity after five transfers on agar medium. The experiments resulted in significant improvement of the studied industrial strains, more than doubling activity of the target enzymes.     

Various <Emphasis Type="Italic">Wolbachia</Emphasis> genotypes differently influence host <Emphasis Type="Italic">Drosophila</Emphasis> dopamine metabolism and survival under heat stress conditions

Background

One of the most widespread prokaryotic symbionts of invertebrates is the intracellular bacteria of Wolbachia genus which can be found in about 50% of insect species. Wolbachia causes both parasitic and mutualistic effects on its host that include manipulating the host reproductive systems in order to increase their transmission through the female germline, and increasing the host fitness. One of the mechanisms, promoting adaptation in biological organisms, is a non-specific neuroendocrine stress reaction. In insects, this reaction includes catecholamines, dopamine, serotonin and octopamine, which act as neurotransmitters, neuromodulators and neurohormones. The level of dopamine metabolism correlates with heat stress resistance in Drosophila adults.

Results

To examine Wolbachia effect on Drosophila survival under heat stress and dopamine metabolism we used five strains carrying the nuclear background of interbred Bi90 strain and cytoplasmic backgrounds with different genotype variants of Wolbachia (produced by 20 backcrosses of Bi90 males with appropriate source of Wolbachia). Non-infected Bi90 strain (treated with tetracycline for 3 generations) was used as a control group. We demonstrated that two of five investigated Wolbachia variants promote changes in Drosophila heat stress resistance and activity of enzymes that produce and degrade dopamine, alkaline phosphatase and dopamine-dependent arylalkylamine N-acetyltransferase. What is especially interesting, wMelCS genotype of Wolbachia increases stress resistance and the intensity of dopamine metabolism, whereas wMelPop strain decreases them. wMel, wMel2 and wMel4 genotypes of Wolbachia do not show any effect on the survival under heat stress or dopamine metabolism. L-DOPA treatment, known to increase the dopamine content in Drosophila, levels the difference in survival under heat stress between all studied groups.

Conclusions

The genotype of symbiont determines the effect that the symbiont has on the stress resistance of the host insect.

     

A sucrose non-fermenting-1-related protein kinase 1 gene from potato, <Emphasis Type="Italic">StSnRK1</Emphasis>, regulates carbohydrate metabolism in transgenic tobacco

Sucrose non-fermenting-1-related protein kinase 1 (SnRK1) has been shown to play an essential role in regulating saccharide metabolism and starch biosynthesis of plant. The regulatory role of StSnRK1 from potato in regulating carbohydrate metabolism and starch accumulation has not been investigated. In this work, a cDNA encoding the SnRK1 protein, named StSnRK1, was isolated from potato. The open reading frame contained 1545 nucleotides encoding 514 amino acids. Subcellular localization analysis in onion epidermal cells indicated that StSnRK1 protein was localized to the nucleus. The coding region of StSnRK1 was cloned into a binary vector under the control of 35S promoter and then transformed into tobacco to obtain transgenic plants. Transgenic tobacco plants expressing StSnRK1 were shown to have a significant increased accumulation of starch content, as well as sucrose, glucose and fructose content. Real-time quantitative PCR analysis indicated that overexpression of StSnRK1 up-regulated the expression of sucrose synthase (NtSUS), ADP-glucose pyrophosphorylase (NtAGPase) and soluble starch synthase (NtSSS III) genes involved in starch biosynthesis in the transgenic plants. In contrast, the expression of sucrose phosphate synthase (NtSPS) gene was decreased in the transgenic plants. Meanwhile, enzymatic analyses indicated that the activities of major enzymes (SUS, AGPase and SSS) involved in the starch biosynthesis were enhanced, whereas SPS activity was decreased in the transgenic plants compared to the wild-type. These results suggest that the manipulation of StSnRK1 expression might be used for improving quality of plants in the future.     

Polymorphism in promoter regions of matrix metalloproteinases (<Emphasis Type="Italic">MMP1</Emphasis>, <Emphasis Type="Italic">MMP9</Emphasis>, and <Emphasis Type="Italic">MMP12</Emphasis>) in chronic obstructive pulmonary disease patients

Our studies have shown that the genotype and allele frequencies of polymorphisms G(?1607)GG of MMP1 gene, C(?1562)T of MMP9 gene, and A(?82)G of MMP12 gene do not significantly differ in the samples of chronic obstructive pulmonary disease (COPD) patients (N = 318) and healthy controls (N = 319) dwelling in Bashkortostan Republic. However, association of (?1562)T allele of the MMP9 gene with the severity of COPD disease progression has been revealed. In COPD patients at stage 4 of the disease, the frequency of allele T was significantly higher that in patients with the stages 2 and 3 (15.89% versus 8.38%; χ² = 7.804; d.f. = 1; P = 0.005; OR = 2.06 95% CI 1.22–3.49). The distribution of the genotype frequencies of C(?1562)T polymorphism of MMP9 gene significantly differed between the patients with various COPD severity (χ² = 9.849; d.f. = 2; P = 0.007). The individuals with rare genotype TT were revealed only among patients with severe COPD form (3.97% versus 0%; χ² = 4.78; P = 0.029; P _cor = 0.058). Analysis of this polymorphism in patients with early COPD onset (younger than 55 years old) has shown a significant increase in the allele T frequency in the group of patients with severe COPD (stage 4 according to GOLD) compared to the patients of the same age but with less severe COPD progression (χ² = 5.26; d.f. = 1; P = 0.022). As the major clinical characteristics of stage 4 COPD is the development of pulmonary emphysema as well as bronchial walls deformation, we suggest that the increased expression of MMP9 gene caused by genetic polymorphism in the gene promoter is important in the early development of serious complications of the disease.     

Constitutive expression of <Emphasis Type="Italic">SlTrxF</Emphasis> increases starch content in transgenic <Emphasis Type="Italic">Arabidopsis</Emphasis>

The plastidic thioredoxin F-type (TrxF) protein plays an important role in plant saccharide metabolism. In this study, a gene encoding the TrxF protein, named SlTrxF, was isolated from tomato. The coding region of SlTrxF was cloned into a binary vector under the control of 35S promoter and then transformed into Arabidopsis thaliana. The transgenic Arabidopsis plants exhibited increased starch accumulation compared to the wild-type (WT). Real-time quantitative PCR analysis showed that constitutive expression of SlTrxF up-regulated the expression of ADP-glucose pyrophosphorylase (AGPase) small subunit (AtAGPase-S1 and AtAGPase-S2), AGPase large subunit (AtAGPase-L1 and AtAGPase-L2) and soluble starch synthase (AtSSS I, AtSSS II, AtSSS III and AtSSS IV) genes involved in starch biosynthesis in the transgenic Arabidopsis plants. Meanwhile, enzymatic analyses showed that the major enzymes (AGPase and SSS) involved in the starch biosynthesis exhibited higher activities in the transgenic plants compared to WT. These results suggest that SlTrxF may improve starch content of Arabidopsis by regulating the expression of the related genes and increasing the activities of the major enzymes involved in starch biosynthesis.     

Engineering of <Emphasis Type="Italic">Escherichia coli</Emphasis> for the synthesis of <Emphasis Type="Italic">N</Emphasis>-hydroxycinnamoyl tryptamine and serotonin

Plants synthesize various phenol amides. Among them, hydroxycinnamoyl (HC) tryptamines and serotonins exhibit antioxidant, anti-inflammatory, and anti-atherogenic activities. We synthesized HC–tryptamines and HC–serotonin from several HCs and either tryptamine or serotonin using Escherichia coli harboring the 4CL (4-coumaroyl CoA ligase) and CaHCTT [hydroxycinnamoyl-coenzyme A:serotonin N-(hydroxycinnamoyl)transferase] genes. E. coli was engineered to synthesize N-cinnamoyl tryptamine from glucose. TDC (tryptophan decarboxylase) and PAL (phenylalanine ammonia lyase) along with 4CL and CaHCTT were introduced into E. coli and the phenylalanine biosynthetic pathway of E. coli was engineered. Using this strategy, approximately 110.6 mg/L of N-cinnamoyl tryptamine was synthesized. By feeding 100 μM serotonin into the E. coli culture, which could induce the synthesis of cinnamic acid or p-coumaric acid, more than 99 μM of N-cinnamoyl serotonin and N-(p-coumaroyl) serotonin were synthesized.     

Enhancement of starch content by constitutive expression of <Emphasis Type="Italic">GmTrxF</Emphasis> in transgenic <Emphasis Type="Italic">Arabidopsis</Emphasis>

The plastidic thioredoxin F-type (TrxF) protein plays an important role in plant carbohydrate metabolism biosynthesis. In this study, a gene encoding the TrxF protein, named GmTrxF, was isolated from soybean. The open reading frame (ORF) contained 540 nucleotides encoding 179 amino acids. The coding region of GmTrxF was cloned into a binary vector under the control of 35S promoter and then transformed into Arabidopsis. The starch content in GmTrxF expressing plants was increased by 57–109% compared to that in wild-type (WT). Real-time quantitative PCR (qRT-PCR) analysis showed that constitutive expression of GmTrxF up-regulated the expression of phosphoglucomutase (AtPGM), ADP-glucose pyrophosphorylase (AGPase) small subunit (AtAGPase-S1 and AtAGPase-S2), AGPase large subunit (AtAGPase-L1 and AtAGPase-L2) and soluble starch synthases (AtSSS I, AtSSS II, AtSSS III and AtSSS IV) genes involved in starch biosynthesis in the transgenic Arabidopsis plants. Meanwhile, enzymatic analyses showed that the major enzymes (AGPase and SSS) involved in the starch biosynthesis exhibited higher activities in the transgenic plants compared to WT. These results suggest that GmTrxF may improve starch content of Arabidopsis by up-regulating the expression of the related genes and increasing the activities of the major enzymes invovled in starch biosynthesis. The manipulation of GmTrxF expression might be used for increasing starch accumulation of plants in the future.     

Effective to census population size ratios in two Near Threatened Mediterranean amphibians: <Emphasis Type="Italic">Pleurodeles waltl</Emphasis> and <Emphasis Type="Italic">Pelobates cultripes</Emphasis>

Efforts to mitigate amphibian declines are hindered by a lack of information about basic aspects of their biology and demography. The effective to census population size ratio (N _e /N _c ) is one of the most important parameters for the management of wildlife populations because it combines information on population abundance and genetic diversity and helps predict population viability in the long term. Few studies have calculated this ratio in amphibians, which sometimes show low ratios, associated with a higher extinction risk. Here we integrate field-based (capture-mark-recapture studies, egg string counts) and molecular approaches (estimation of the effective number of breeders (N _b ) and the effective population size (N _e ) based on genotypes from larval cohorts and candidate parents) to produce the first estimates of the N _e /N _c and N _b /N _c ratios in two amphibians, the Iberian ribbed newt Pleurodeles waltl and the western spadefoot Pelobates cultripes. Additionally, we investigate sex-biased dispersal in both species based on direct (field observations) and indirect (genetic) evidence. Both species showed similar ratios, slightly lower in Pleurodeles (0.21–0.24) than in Pelobates (0.25–0.30). Observed displacement rates were low in both species (P. waltl?=?0.51%; P. cultripes?=?1.23%). We found no evidence for sex-biased dispersal in P. cultripes, but both direct and indirect evidences suggest a tendency for female-biased dispersal in P. waltl. We discuss differences in the genetic estimates of N _e and N _b provided by three inference methods and the implications of our findings for the management of these species, characteristic of Mediterranean wetlands in the Iberian Peninsula and listed as Near Threatened.