Isozyme Genes Research Articles

Identification and characterization of the critical genes encoding Cd-induced enhancement of SOD isozymes activities in Zhe-Maidong (Ophiopogon japonicus).

Superoxide dismutase (SOD) protects plants from abiotic stress-induced reactive oxygen species (ROS) damage. Here, the effects of cadmium (Cd) exposure on ROS accumulation and SOD isozymes, as well as the identification of significant SOD isozyme genes, were investigated under different Cd stress treatments to Zhe-Maidong (Ophiopogon japonicus). The exposure to Cd stress resulted in a notable elevation in the SOD activity in roots. Cu/ZnSODa and Cu/ZnSODb were the most critical SOD isozymes in response to Cd stress, as indicated by the detection results for SOD isozymes. A total of 22 OjSOD genes were identified and classified into three subgroups, including 10 OjCu/ZnSODs, 6 OjMnSODs, and 6 OjFeSODs, based on the analysis of conserved motif and phylogenetic tree. Cu/ZnSOD-15, Cu/ZnSOD-18, Cu/ZnSOD-20, and Cu/ZnSOD-22 were the main genes that control the increase in SOD activity under Cd stress, as revealed via quantitative PCR and transcriptome analysis. Additionally, under various heavy metal stress (Cu2+, Fe2+, Zn2+, Mn2+), Cu/ZnSOD-15, Cu/ZnSOD-18, and Cu/ZnSOD-22 gene expression were significantly upregulated, indicating that these three genes play a critical part in resisting heavy metal stress. The molecular docking experiments performed on the interaction between oxygen ion (O2•-) and OjSOD protein have revealed that the critical amino acid residues involved in the binding of Cu/ZnSOD-22 to the substrate were Pro135, Ile136, Ile140, and Arg144. Our findings provide a solid foundation for additional functional investigations on the OjSOD genes, as well as suggestions for improving genetic breeding and agricultural management strategies to increase Cd resistance in O. japonicus.

Upregulation of Phospholipase C Gene Expression Due to Norepinephrine-Induced Hypertrophic Response.

The activation of phospholipase C (PLC) is thought to have a key role in the cardiomyocyte response to several different hypertrophic agents such as norepinephrine, angiotensin II and endothelin-1. PLC activity results in the generation of diacylglycerol and inositol trisphosphate, which are downstream signal transducers for the expression of fetal genes, increased protein synthesis, and subsequent cardiomyocyte growth. In this article, we describe the signal transduction elements that regulate PLC gene expression. The discussion is focused on the norepinephrine- α1-adrenoceptor signaling pathway and downstream signaling processes that mediate an upregulation of PLC isozyme gene expression. Evidence is also indicated to demonstrate that PLC activities self-regulate the expression of PLC isozymes with the suggestion that PLC activities may be part of a coordinated signaling process for the perpetuation of cardiac hypertrophy. Accordingly, from the information provided, it is plausible that specific PLC isozymes could be targeted for the mitigation of cardiac hypertrophy.

Features of Picea abies (L.) Karst. population structure formation on the territory of Belarus

One of the main forest forming tree species in Belarus is the European spruce (Picea abies (L.) Karst.). The formation of European spruce forest population genetic structure took place under the influence of migration flows from different refugia during the postglacial period. For the genogeographic study of P. abies 25 isozyme genes (Aat-1, Aat-2, Adh, Gdh, Idh-1, Idh-2, Mdh-1, Mdh-2, Mdh-3, Skdh, 6-Pgd-1, 6-Pgd-2, 6-Pgd-3, Lap-1, Lap-2, Sdh, Gpi, Hk, Me, Dia-1, Dia-2, Dia-4, Pgm-1, Pgm-2, Fl-Est) of nuclear DNA (analysis was carried out in 10 populations), 3 microsatellite loci (Pt63718, Pt26081, Pt71936) of chloroplast DNA (57 populations were considered) and 1 microsatellite locus (mt15-D02) of mitochondrial DNA (56 populations were studied) were used. As a result, 82 allelic variants of isozyme genes, 19 allelic variants of chloroplast DNA loci and 2 allelic variants of mitochondrial DNA locus were found. The spatial distribution of the alleles was defined and the regional features of the genogeographic differentiation of the spruce forest were considered. The presence of two migration flows representatives (southern and northern) in Belarus was confirmed. It was shown that the highest concentration of P. abies trees with southern (Carpathian) origin is observed in the southwest of the country. Clinal variability was revealed for a number of markers in the directions from south to north and from west to east. In general the data obtained are consistent with the results of studies based on the analysis of the spatial distribution of the cone scales traits.

English

The new soybean stay-green variety Jinda Zhilv No. 1 (Z1) was obtained through crossing a stay-green mutant with the super-high yielding soybean cultivar Jinda No. 74 (JD74). Here, we compared the antioxidant enzyme activities and reactive oxygen species content of the Z1 and JD74 varieties under natural and dark-induced senescence. Dark treatment was imposed at the seedling stage for 13 days. Fluorescence quantitative PCR was used to investigate the expression of isozyme genes related to superoxide dismutase (SOD), catalase (CAT) and ascorbate–glutathione cycle. The results indicated that compared with JD74, Z1 exhibited enhanced antioxidant enzyme activity, with rates of hydrogen peroxide and superoxide anion accumulation being lower in Z1 after flowering. The expression levels of antioxidant enzyme isogenes, including Mn-SOD, Chl Cu/Zn-SOD, peroxisome Cu/Zn-SOD, CAT5, MDHAR1, and DHAR3, were higher in Z1 than in JD74 during the seed-filling stage. After 6 days of dark treatment, the membrane system of JD74 leaves showed severe oxidative damage and the leaves had turned completely yellow. These changes were accompanied by reduced contents of chlorophyll and soluble protein after 13 days of dark treatment. In contrast, Z1 was observed to be more tolerant to dark stress. Its internal reactive oxygen metabolism balance remained unimpaired, and the leaves showed no obvious senescence traits. In conclusion, the higher antioxidant capacity in Z1 contributes to delayed leaf senescence, which is a significant finding with respect to the application of stay-green mutants in soybean breeding and germplasm innovation. © 2021 Friends Science Publishers

Benzo(a)pyrene, an Active Product of Cigarette Smoke, Role in PLA2 Isoforms Activation in Colon Cancer

Background and aim: One of the active combustion product of cigarette smoke, Benzo[a]pyrenes, role in pulmonary cancer is clearly understood. However, its role in gastrointestinal cancer including colon cancer is not clearly understood. Methods: In this study, benzo(a)pyrenes was treated to colon cells to evaluate its role in cell viability, cellular ROS, and gene expression of various PLA2 isoforms was evaluated by FACS and PCR. The identified PLA2 was silenced at the gene level to evaluate its role in cell viability and ROS generation. Results: B(a)P treatment at 1 µg/mL for 48 h to HCT-15 male colon cells significantly reduced the cell viability without affecting HT-29 female colon cells. Higher doses and longer treatment duration with B(a)P showed that female colon cells were highly sensitive than male colon cells. Annexin-V/PI staining for preapoptotic detection showed that B(a)P treatment increased the apoptosis in both the cell types in a concentration and time-dependent manner. The cytosolic ROS (cROS) and superoxide radical (SOR) formation in the female colon cells was significantly higher than male colon cells unlike the mitochondrial ROS (mtROS) production which was significantly higher in male colon cells. Treatment with B(a)P significantly upregulated the IID and IVA PLA2 isoform groups in HCT-15 male colon cells, whereas IB was upregulated in HT-29 female colon cells among the various PLA2 isozyme gene studied (IB, IID, III, IVA, IVB, IVC, VI, X, aiPLA2 and iPLA2). Gene silencing experiments targeting PLA2 IID and IVA in the HCT-15 male colon cells and IB in HT-29 female colon cells showed no effect with B(a)P treatment on the cell proliferation, apoptosis, membrane integrity and free radicals (ROS, mtROS, and SOR) generation. Conclusion: Targeting specific PLA2 isozymes in a cell-specific manner abolished the B(a)P-induced PLA2-mediated oxidative damage–related signaling pathways.

PO-239 Benzo(a)pyrene, an active product of cigarette smoke, role in PLA2 isoforms activation in colon cancer

IntroductionOne of the active combustion product of cigarette smoke, Benzo[a]pyrenes, role in pulmonary cancer is clearly understood. However, its role in gastrointestinal cancer including colon cancer is not clearly understood.Material and methodsIn this study, benzo(a)pyrene’s was treated to colon cells to evaluate its role in cell viability, cellular ROS, and gene expression of various PLA2 isoforms was evaluated by FACS and PCR. The identified PLA2 was silenced at the gene level to evaluate its role in cell viability and ROS generation.Results and discussionsB(a)P treatment at 1 µg/ml for 48 hour to HCT-15 male colon cells significantly reduced the cell viability without affecting HT-29 female colon cells. Higher doses and longer treatment duration with B(a)P showed that female colon cells were highly sensitive than male colon cells. Annexin-V/PI staining for pre-apoptotic detection showed that B(a)P treatment increased the apoptosis in both the cell types in a concentration and time-dependent manner. The cytosolic ROS (cROS) and superoxide radical (SOR) formation in the female colon cells was significantly higher than male colon cells unlike the mitochondrial ROS (mtROS) production which was significantly higher in male colon cells. Treatment with B(a)P significantly upregulated the IID and IVA PLA2 isoform groups in HCT-15 male colon cells, whereas IB was upregulated in HT-29 female colon cells among the various PLA2 isozyme gene studied (IB, IID, III, IVA, IVB, IVC, VI, X, aiPLA2 and iPLA2). Gene silencing experiments targeting PLA2 IID and IVA in the HCT-15 male colon cells and IB in HT-29 female colon cells showed no effect with B(a)P treatment on the cell proliferation, apoptosis, membrane integrity and free radicals (ROS, mtROS, and SOR) generation.ConclusionTargeting specific PLA2 isozymes in a cell specific manner abolished the B(a)P-induced PLA2 mediated oxidative damage related signalling pathways.

The FBH family of bHLH transcription factors controls ACC synthase expression in sugarcane.

Ethylene is a phytohormone involved in the regulation of several aspects of plant development and in responses to biotic and abiotic stress. The effects of exogenous application of ethylene to sugarcane plants are well characterized as growth inhibition of immature internodes and stimulation of sucrose accumulation. However, the molecular network underlying the control of ethylene biosynthesis in sugarcane remains largely unknown. The chemical reaction catalyzed by 1-aminocyclopropane-1-carboxylic acid synthase (ACS) is an important rate-limiting step that regulates ethylene production in plants. In this work, using a yeast one-hybrid approach, we identified three basic helix-loop-helix (bHLH) transcription factors, homologs of Arabidopsis FBH (FLOWERING BHLH), that bind to the promoter of ScACS2 (Sugarcane ACS2), a sugarcane type 3 ACS isozyme gene. Protein-protein interaction assays showed that sugarcane FBH1 (ScFBH1), ScFBH2, and ScFBH3 form homo- and heterodimers in the nucleus. Gene expression analysis revealed that ScFBHs and ScACS2 transcripts are more abundant in maturing internodes during afternoon and night. In addition, Arabidopsis functional analysis demonstrated that FBH controls ethylene production by regulating transcript levels of ACS7, a homolog of ScACS2. These results indicate that ScFBHs transcriptionally regulate ethylene biosynthesis in maturing internodes of sugarcane.

Molecular diversity of tuliposide B-converting enzyme in tulip (Tulipa gesneriana): identification of the root-specific isozyme.

6-Tuliposide B (PosB) is a glucose ester accumulated in tulip (Tulipa gesneriana) as a major secondary metabolite. PosB serves as the precursor of the antimicrobial lactone tulipalin B (PaB), which is formed by PosB-converting enzyme (TCEB). The gene TgTCEB1, encoding a TCEB, is transcribed in tulip pollen but scarcely transcribed in other tissues (e.g. roots) even though those tissues show high TCEB activity. This led to the prediction of the presence of a TCEB isozyme with distinct tissue specificity. Herein, we describe the identification of the TgTCEB-R gene from roots via native enzyme purification; this gene is a paralog of TgTCEB1. Recombinant enzyme characterization verified that TgTCEB-R encodes a TCEB. Moreover, TgTCEB-R was localized in tulip plastids, as found for pollen TgTCEB1. TgTCEB-R is transcribed almost exclusively in roots, indicating a tissue preference for the transcription of TCEB isozyme genes.

遺伝子発現の調節機構 : (1)大腸菌divE42変異株におけるlacZ遺伝子発現の調節機構 (2)酵母菌Saccharomyces cerevisiaeのフマル酸還元酵素アイソザイム遺伝子発現の調節(平成8年度プロジェクト研究報告(保健学部・保健学研究科),研究助成金による成果紹介)

遺伝子発現の調節機構 : (1)大腸菌divE42変異株におけるlacZ遺伝子発現の調節機構 (2)酵母菌Saccharomyces cerevisiaeのフマル酸還元酵素アイソザイム遺伝子発現の調節(平成8年度プロジェクト研究報告(保健学部・保健学研究科),研究助成金による成果紹介)

Tissue distribution and early developmental expression patterns of aldolase A, B, and C in grass carp Ctenopharyngodon idellus.

Aldolase is a key enzyme involved in glycolysis, gluconeogenesis, and the pentose phosphate pathway. To establish the expression patterns of all three aldolase isozyme genes in different tissues and during early embryogenesis in lower vertebrates, as well as to explore the functional differences between these three isozymes, the grass carp was selected as a model owing to its relatively high glucose-metabolizing capability. Based on the cDNA sequences of the aldolase A, B, and C genes, the expression patterns of these three isozymes were analyzed in different tissues and during early embryogenesis using quantitative real-time polymerase chain reaction (qRT-PCR). Sequence analysis of cDNAs indicated that aldolase A, B, and C (GenBank accession numbers: KM192250, KM192251, and KM192252) consist of 364, 364, and 363 amino acids, respectively. The qRT-PCR results showed that the expression levels of aldolase A, B, and C were highest in the muscle, liver, and brain, respectively. Aldolase A and C exhibited similar expression patterns during embryogenesis, with high levels observed in unfertilized and fertilized eggs and at the blastocyst stage, followed by a decline and then increase after organogenesis. In contrast, aldolase B transcript was not detected during the unfertilized egg stage, and appeared only from gastrulation; the expression increased markedly during the feeding period (72 h after hatching), at which point the level was higher than those of aldolase A and C. These data suggest that the glucose content of grass carp starter feed should be adjusted according to the metabolic activity of aldolase B.

Effects of overexpression of PKAc genes on expressions of lignin-modifying enzymes by Pleurotus ostreatus.

We studied the role of genes encoding the cAMP-dependent protein kinase A catalytic subunit (PKAc) in the ligninolytic system in Pleurotus ostreatus. The wild-type P. ostreatus strain PC9 has two PKAc-encoding genes: PKAc1 and PKAc2 (protein ID 114122 and 85056). In the current study, PKAc1 and PKAc2 were fused with a β-tubulin promoter and introduced into strain PC9 to produce the overexpression strains PKAc1-97 and PKAc2-69. These strains showed significantly higher transcription levels of isozyme genes encoding lignin-modifying enzymes than strain PC9, but the specific gene expression patterns differed between the two recombinant strains. Both recombinants showed 2.05-2.10-fold faster degradation of beechwood lignin than strain PC9. These results indicate that PKAc plays an important role in inducing the wood degradation system in P. ostreatus.

Transcriptional Profiles of Hybrid Eucalyptus Genotypes with Contrasting Lignin Content Reveal That Monolignol Biosynthesis-related Genes Regulate Wood Composition.

Eucalyptus species constitutes the most widely planted hardwood trees in temperate and subtropical regions. In this study, we compared the transcript levels of genes involved in lignocellulose formation such as cellulose, hemicellulose and lignin biosynthesis in two selected 3-year old hybrid Eucalyptus (Eucalyptus urophylla × Eucalyptus grandis) genotypes (AM063 and AM380) that have different lignin content. AM063 and AM380 had 20.2 and 35.5% of Klason lignin content and 59.0 and 48.2%, α-cellulose contents, respectively. We investigated the correlation between wood properties and transcript levels of wood formation-related genes using RNA-seq with total RNAs extracted from developing xylem tissues at a breast height. Transcript levels of cell wall construction genes such as cellulose synthase (CesA) and sucrose synthase (SUSY) were almost the same in both genotypes. However, AM063 exhibited higher transcript levels of UDP-glucose pyrophosphorylase and xyloglucan endotransglucoxylase than those in AM380. Most monolignol biosynthesis-related isozyme genes showed higher transcript levels in AM380. These results indicate monolignol biosynthesis-related genes may regulate wood composition in Eucalyptus. Flavonoids contents were also observed at much higher levels in AM380 as a result of the elevated transcript levels of common phenylpropanoid pathway genes, phenylalanine ammonium lyase, cinnamate-4-hydroxylase (C4H) and 4-coumarate-CoA ligase (4CL). Secondary plant cell wall formation is regulated by many transcription factors. We analyzed genes encoding NAC, WRKY, AP2/ERF, and KNOX transcription factors and found higher transcript levels of these genes in AM380. We also observed increased transcription of some MYB and LIM domain transcription factors in AM380 compared to AM063. All these results show that genes related to monolignol biosynthesis may regulate the wood composition and help maintain the ratio of cellulose and lignin contents in Eucalyptus plants.

Interisland variegation of venom [Lys49]phospholipase A2 isozyme genes in Protobothrops genus snakes in the southwestern islands of Japan

Protobothrops tokarensis (Pt), a Crotalinae snake, inhabits only Takarajima and Kodakarajima islands of the Tokara Islands located in the immediate north of Amami-Oshima island of Japan. Kodakarajima P. tokarensis venom gland cDNA library gave four types of phospholipase A2 (PLA2) cDNAs encoding neutral [Asp49]PLA2, basic [Asp49]PLA2, highly basic [Asp49]PLA2, and [Lys49]PLA2. As the amino acid sequences encoded by their open reading frames (ORFs) were identical to those of PLA2, PLA-B, PLA-N, and BPI (a [Lys49]PLA2), respectively, from Amami-Oshima P. flavoviridis (Pf) venom, they were named PtPLA2, PtPLA-B, PtPLA-N, and PtBPI. Chromatography of P. tokarensis venom gave three PLA2 isozymes, PtPLA2, PtPLA-B, and PtBPI. However, BPII and BPIII ([Lys49]PLA2s) expressed in Amami-Oshima P. flavoviridis venom were not found in P. tokarensis venom. Genomic polymerase chain reaction (PCR) for P. tokarensis liver DNAs with the unique primers gave PtBPI gene. Notably it was found that LINE (long interspersed nuclear element)-1 fragment is inserted into second intron of PtBPI gene. The LINE-1 fragment may prevent duplication of PtBPI gene and thus formation of plural [Lys49]PLA2 genes in P. tokarensis genome. The interisland variegation of venom [Lys49]PLA2 isozyme genes in Protobothrops genus snakes in the southwestern islands of Japan is discussed.

Characterization of the superoxide dismutase genes of the halophyte Suaeda maritima in Japan and Egypt.

Suaeda maritima varieties native to Japan and Egypt were cultured under aseptic conditions. The varieties differed in genetic distance but exhibited similar expression profiles of superoxide dismutase isozyme genes. The expression characteristics of superoxide dismutase (SOD; EC 1.15.1.1) isozyme genes from halophytic Suaeda marit ima plants native to Japan and Egypt were analyzed using young plants grown under aseptic conditions. A phylogenetic tree based on internal transcribed spacer sequences suggested that Egyptian S. maritima is related to European and India S. maritima, while Japanese S. maritima belongs to a separate clade. An in-gel SOD activity staining assay revealed that leaves from both the Egyptian and Japanese varieties showed high levels of CuZn-SOD and Fe-SOD activity, but no Mn-SOD activity; conversely, stems from both varieties showed Mn-SOD activity as well as other SOD isozyme activities. In Japanese S. maritima leaves, SOD activity was increased by incubation in growth medium containing 400 mM NaCl, while Egyptian S. maritima leaves showed elevated SOD activity in the absence of high salt. Genes encoding Mn-SOD and Fe-SOD were isolated from both plant types. RT-PCR analysis revealed that all SOD isozyme-encoding genes were expressed at the same levels in leaves from both plant types grown in normal or high-salt medium. In contrast, the expression of genes encoding choline monooxygenase and betaine aldehyde dehydrogenase, which are involved in betacyanin biosynthesis, was increased in high-salt medium. In leaves of Japanese S. maritima plants, Fe deficiency without high salt exposure preferentially decreased Fe-SOD activity. On the other hand, Fe deficiency with high salt exposure decreased not only Fe-SOD activity but also CuZn-SOD activity, suggesting that Fe availability is involved in the up-regulation of SOD isozymes mediating salt tolerance.

Selection of suitably non-repressing carbon sources for expression of alcohol oxidase isozyme promoters in the methylotrophic yeast Pichia methanolica

In this work, we aimed to select suitable non-repressing carbon sources for the expression of promoters derived from the alcohol oxidase (AOD) isozyme genes, PMOD1 and PMOD2, during the growth of Pichia methanolica. Our results revealed that xylose is the best non-repressing carbon source for heterologous gene expression using both PMOD1 and PMOD2, and that glycerol is also a suitable carbon source with by which the on/off of PMOD2 expression can be controlled.

The finding of a group IIE phospholipase A2 gene in a specified segment of Protobothrops flavoviridis genome and its possible evolutionary relationship to group IIA phospholipase A2 genes.

The genes encoding group IIE phospholipase A2, abbreviated as IIE PLA2, and its 5' and 3' flanking regions of Crotalinae snakes such as Protobothrops flavoviridis, P. tokarensis, P. elegans, and Ovophis okinavensis, were found and sequenced. The genes consisted of four exons and three introns and coded for 22 or 24 amino acid residues of the signal peptides and 134 amino acid residues of the mature proteins. These IIE PLA2s show high similarity to those from mammals and Colubridae snakes. The high expression level of IIE PLA2s in Crotalinae venom glands suggests that they should work as venomous proteins. The blast analysis indicated that the gene encoding OTUD3, which is ovarian tumor domain-containing protein 3, is located in the 3' downstream of IIE PLA2 gene. Moreover, a group IIA PLA2 gene was found in the 5' upstream of IIE PLA2 gene linked to the OTUD3 gene (OTUD3) in the P. flavoviridis genome. It became evident that the specified arrangement of IIA PLA2 gene, IIE PLA2 gene, and OTUD3 in this order is common in the genomes of humans to snakes. The present finding that the genes encoding various secretory PLA2s form a cluster in the genomes of humans to birds is closely related to the previous finding that six venom PLA2 isozyme genes are densely clustered in the so-called NIS-1 fragment of the P. flavoviridis genome. It is also suggested that venom IIA PLA2 genes may be evolutionarily derived from the IIE PLA2 gene.

Increases in intracellular calcium via activation of potentially multiple phospholipase C isozymes in mouse olfactory neurons.

Phospholipase C (PLC) and internal Ca2+ stores are involved in a variety of cellular functions. However, our understanding of PLC in mammalian olfactory sensory neurons (OSNs) is generally limited to its controversial role in odor transduction. Here we employed single-cell Ca2+ imaging and molecular approaches to investigate PLC-mediated Ca2+ responses and its isozyme gene transcript expression. We found that the pan-PLC activator m-3M3FBS (25 μM) induces intracellular Ca2+ increases in vast majority of isolated mouse OSNs tested. Both the response amplitude and percent responding cells depend on m-3M3FBS concentrations. In contrast, the inactive analog o-3M3FBS fails to induce Ca2+ responses. The m-3M3FBS-induced Ca2+ increase is blocked by the PLC inhibitor U73122, while its inactive analog U73433 has no effect. Removal of extracellular Ca2+ does not change significantly the m-3M3FBS-induced Ca2+ response amplitude. Additionally, in the absence of external Ca2+, we found that a subset of OSNs respond to an odorant mixture with small Ca2+ increases, which are significantly suppressed by U73122. Furthermore, using reverse transcription polymerase chain reaction and real-time quantitative polymerase chain reaction, we found that multiple PLC isozyme gene transcripts are expressed in olfactory turbinate tissue in various levels. Using RNA in situ hybridization analysis, we further show expression of β4, γ1, γ2 gene transcripts in OSNs. Taken together, our results establish that PLC isozymes are potent enzymes for mobilizing intracellular Ca2+ in mouse OSNs and provide molecular insight for PLC isozymes-mediated complex cell signaling and regulation in the peripheral olfactory epithelium.

Module-based functional pathway enrichment analysis of a protein-protein interaction network to study the effects of intestinal microbiota depletion in mice

Complex communities of microorganisms play important roles in human health, and alterations in the intestinal microbiota may induce intestinal inflammation and numerous diseases. The purpose of this study was to identify the key genes and processes affected by depletion of the intestinal microbiota in a murine model. The Affymetrix microarray dataset GSE22648 was downloaded from the Gene Expression Omnibus database, and differentially expressed genes (DEGs) were identified using the limma package in R. A protein-protein interaction (PPI) network was constructed for the DEGs using the Cytoscape software, and the network was divided into several modules using the MCODE plugin. Furthermore, the modules were functionally annotated using the PiNGO plugin, and DEG-related pathways were retrieved and analyzed using the GenMAPP software. A total of 53 DEGs were identified, of which 26 were upregulated and 27 were downregulated. The PPI network of these DEGs comprised 3 modules. The most significant module-related DEGs were the cytochrome P450 (CYP) 4B1 isozyme gene (CYP4B1) in module 1, CYP4F14 in module 2 and the tachykinin precursor 1 gene (TAC1) in module 3. The majority of enriched pathways of module 1 and 2 were oxidation reduction pathways (metabolism of xenobiotics by CYPs) and lipid metabolism-related pathways, including linoleic acid and arachidonic acid metabolism. The neuropeptide signaling pathway was the most significantly enriched functional pathway of module 3. In conclusion, our findings strongly suggest that intestinal microbiota depletion affects cellular metabolism and oxidation reduction pathways. In addition, this is the first time, to the best of our knowledge, that the neuropeptide signaling pathway is reported to be affected by intestinal microbiota depletion in mice. The present study provides a list of candidate genes and processes related to the interaction of microbiota with the intestinal tract.

Cold acclimation induces freezing tolerance via antioxidative enzymes, proline metabolism and gene expression changes in two chrysanthemum species

Cold acclimation is necessary for chrysanthemum to achieve its genetically determined maximum freezing tolerance, but the underlying physiological and molecular mechanisms are unclear. The aim of this study was to discover whether changes in antioxidative enzymes, proline metabolism and frost-related gene expression induced by cold acclimation are related to freezing tolerance. Our results showed that the semi-lethal temperature (LT50) decreased from -7.3 to -23.5°C in Chrysanthemum dichrum and -2.1 to -7.1°C in Chrysanthemum makinoi, respectively, after cold acclimation for 21days. The activities of SOD, CAT and APX showed a rapid and transient increase in the two chrysanthemum species after 1day of cold acclimation, followed by a gradual increase during the subsequent days and then stabilization. qRT-PCR analysis showed that the expression levels of some isozyme genes (Mn SOD, CAT and APX) were upregulated, which was consistent with the SOD, CAT and APX activities, while others remained relatively constant (Fe SOD and Cu/Zn SOD). P5CS and PDH expression were increased under cold acclimation and the level of P5CS presented similar trends as proline content, indicating proline accumulation was via P5CS and PDH cooperation. Cold acclimation also promoted DREB, COR413 and CSD gene expression. The activities of three enzymes and gene expression were higher in C. dichrum than in C. makinoi after cold acclimation. Our data suggested that cold-inducible freezing-tolerance could be attributed to higher activity of antioxidant enzymes, and increased proline content and frost-related gene expression during different periods.

Analysis of the genetic structure of a model Scots pine (Pinus sylvestris) seed orchard for development of management strategies

Genetic structure, diversity and clonal homogeneity were determined on the basis of the isozyme gene markers in a model Scots pine seed orchard in the north-eastern part of the Czech Republic that originated from natural forest regions NFR 28 and 29 (Jeseníky Mts.) and the 2<sup>nd</sup> and 3<sup>rd</sup> forest altitudinal zones (FAZ). Clone and ramet crop variation for the 2009–2011 period was assessed. Comparing the loci measured also in two pine orchards in central Slovakia, a similar proportion of homogeneous clones but considerably lower expected heterozygosity were found out for most of the measured isozyme loci. Heterozygosity of the investigated orchard did not change considerably after the exclusion of alien and wrongly placed ramets. No difference in average cone production between clones originating from different NFR was observed. Verification of the clone identity of seed orchards managed in a certain way can be suggested with the subsequent removal of alien ramets.