The structure and assembly of rhizobacterial communities are influenced by poplar genotype.

The interaction between plants and microbes dominates plant growth and fitness in specific environments. The study of the relationship between plant genotypes and rhizobacterial community structure would provide a deep insight into the recruitment strategies of plants toward soil bacteria. In this study, three genotypes of 18-year-old mature poplar (H1, H2, and H3) derived from four different parents were selected from a germplasm nursery of Populus deltoides. Rhizosphere soil carbon, nitrogen, and phosphorus properties as well as the 16S rDNA sequences of rhizobacterial communities were analyzed to determine the relationship between poplar genotypes and rhizobacterial communities assembly. The results showed there were significant differences in the diversity (Chao1, ACE index, and Shannon index) of rhizobacterial communities between H1 and H2, as well as between H2 and H3, but no difference between H1 and H3. Principal component analysis also revealed a similar structure of rhizobacterial communities between H1 and H3, whereas the rhizobacterial communities of H2 demonstrated significant differences from H1 and H3. Linear discriminant effect size analysis indicated that there were 11 and 14 different biomarkers in the H1 and H3 genotype, respectively, but 42 in the H2 genotype. Co-occurrence network analysis indicated that the rhizobacterial communities of H2 had a distinct network structure compared to those of the other two genotypes, whereas H1 and H3 had a similar pattern of co-occurrence network. Threshold indicator taxa analysis revealed that 63 genera responded significantly to NO3 --N content and 58 genera to NH4 +-N/NO3 --N ratio. Moreover, the stochastic assembly process was found to be decreased with increasing NO3 --N content and fluctuated with increasing NH4 +-N/NO3 --N ratio. All results indicated that the structure of poplar rhizobacterial communities were influenced by host genotypes, and available nitrogen might play a dominant role in the assembly of rhizobacterial communities. This study would promote the future selection and utilization of rhizobacteria in poplar breeding.

The application of gene splitting technique for controlling transgene flow in rice.

Controlling transgene flow in China is important, as this country is part of the center of origin of rice. A gene-splitting technique based on intein-mediated trans-splicing represents a new strategy for controlling transgene flow via biological measures. In this study, the G2-aroA gene which provides glyphosate tolerance was split into an N-terminal and a C-terminal region, which were then fused to intein N and intein C of the Ssp DnaE intein, ultimately forming EPSPSn:In and Ic:EPSPSc fusion genes, respectively. These fusion genes were subsequently transformed into the rice cultivar Zhonghua 11 via the Agrobacterium-mediated method. The two split gene fragments were then introduced into the same rice genome by genetic crossings. Glyphosate tolerance analysis revealed that the functional target protein was reconstituted by Ssp DnaE intein-mediated trans-splicing and that the resultant hybrid rice was glyphosate tolerant. The reassembly efficiency of the split gene fragments ranged from 67 to 91% at the molecular level, and 100% of the hybrid F1 progeny were glyphosate tolerant. Transgene flow experiments showed that when the split gene fragments are inserted into homologous chromosomes, the gene-splitting technique can completely avoid the escape of the target trait to the environment. This report is the first on the reassembly efficiency and effectiveness of transgene flow containment via gene splitting in rice. This study provides not only a new biological strategy for controlling rice transgene flow but also a new method for cultivating hybrid transgenic rice.

Archaeal community succession with fine root growth in poplar plantation.

The archaeal community structure in the rhizosphere soils might change with root growth, which is of great importance for understanding the interaction between roots and microbes. According to root colors, three groups of rhizosphere soils from first-order fine roots of poplar trees (Populus × euramericana) were sampled, including rhizosphere soils surrounding newly born roots (white color, WR), mature roots (yellow color, YR) and aged roots (brown color, BR). Total microbial DNA was extracted from the soils associated with poplar fine roots. The specific primers were used to amplify the 16S rDNA V4-V5 region of soil archaea, and the Illumina MiSeq platform was used for high-throughput sequencing analysis. The results showed that the observed OTU (operational taxonomic unit) abundance of archaeal community in WR and BR rhizosphere soils were similar, while the OTU abundance in YR rhizosphere soil were lower. The WR and BR shared 134 OTUs of archea, the YR and BR shared 87 OTUs, and the WR and BR shared 90 OTUs. The Chao1 index and the ACE index of archaeal community in YR rhizosphere soil were significantly lower than those of WR and BR, while the Simpson index and the Shannon index of BR were significantly lower than WR to YR. Results from the PERMANOVA analysis showed that archaeal community compositions in WR and BR rhizosphere soils were significantly different. Species annotation showed that there were 12 genera of archea in three rhizosphere soils, five genera in WR, 10 genera in YR, and six genera in BR, respectively. The similarity of the archaeal community composition in poplar rhizosphere soils gradually decreased from WR to BR, with large differences among different growth stages of fine roots. The dominant genus was Candidatus_Nitrososphaera, with a relative abundance of more than 70%, indicating this archaea group might be closely related to poplar fine roots development.

Regulatory effects of root pruning on leaf nutrients, photosynthesis, and growth of trees in a closed-canopy poplar plantation.

A plantation of 5-year-old poplar Populus × euramericana cv. 'Neva' was used to study the regulatory effects of root pruning on nutrients, photosynthetic characteristics, and water-use efficiency (WUE) of leaves and growth rates of diameter at breast height (DBH; 1.3 m), tree height, and volume. Six root-pruning treatments were conducted with different combinations of intensity (at a distance of six, eight or ten times DBH from the trunk) and orientation (on two or four sides of the trees). Results showed that the N, P, K, photosynthetic rate, transpiration rate, and stomatal conductance of leaves were all significantly decreased by root pruning over the initial period following root pruning (30 days), but increased in the subsequent investigations. The values of the above indexes peaked in 8-2 treatment (i.e., eight times DBH distance on two sides). The leaf WUE in 8-2 treatment, and average growth rates of DBH, tree height and volume, were the highest among all treatments within 3 years of root pruning. The results indicated that the root pruning based on the appropriate selection of intensity and orientation had significant positive effects on leaf nutrients, photosynthesis, and growth of trees in a closed-canopy poplar plantation.

Effects of root pruning on the physicochemical properties and microbial activities of poplar rhizosphere soil.

This study aimed to determine the effects of root pruning on the physicochemical characteristics and microbial activities of poplar rhizosphere soil. The root systems of 5-year-old poplar (Populus×euramericana cv. 'Neva') trees were manually pruned at 6, 8, or 10 times diameter at breast height (DBH) from the trunk (severe, moderate, and light, respectively) along both inter-row sides. Moderate root pruning significantly increased the concentrations of amino acids, organic acids, and total sugars in the root exudates and decreased the pH of rhizosphere soil. This treatment also increased the contents of available nitrogen, phosphorus, potassium, and total organic carbon as well as high-, medium-, and low-activity organic carbon in rhizosphere soil. Moreover, moderate pruning increased the contents of microbial biomass carbon and nitrogen, and enhanced basal respiration, in addition to decreasing the metabolic quotients in rhizosphere soil by 8.9%, 5.0%, and 11.4% compared with control, light, and severe root pruning treatments, respectively. Moderate pruning increased the growth rates of DBH, tree height, and volume to the highest levels. Furthermore, these indices were not significantly different between the light root pruning and control groups, but varied significantly between severe and moderate root-pruning treatments. Thus, root pruning, depending on the distance from the trunk, significantly influences the physicochemical properties and microbial activities in poplar rhizosphere soil.

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The study on microbial community composition in rhizosphere soils surrounding different order roots is of great significance for understanding the interactions between roots and microbes. Using Illumina Miseq sequencing technology, this study analyzed the differences of fungal community structure in bulk soils and rhizosphere soils surrounding different root orders of poplar (Populus × euramericana 'Neva') tree. The microbial species annotation showed that 128, 124, 130 and 101 fungal genera were classified in the rhizosphere soils around 1-2 order roots (R1), 3 order roots (R2), 4-5 order roots (R3) and in the bulk soils (NR), respectively. The differences of present fungal genera indicated a selectivity mechanism driving fungal community assembly in poplar rhizosphere soils. There were seven fungal genera with more than 1% of relative abundance in rhizosphere soils. Trichoderma was the dominant fungal genus in R1. Trichosporon and Aspergillus were the dominant fungal genera in R2 and R3, respectively. Alpha (α) diversity indices showed that the fungal diversity was significantly different among root orders. Specifically, the diversity of soil fungal community in the rhizosphere soils around lower order roots was significantly higher than that of higher order roots (P＜0.05). Beta (ß) diversity indices showed that the dissimilarity of fungal community composition increased along with the root orders. All these results implied the different composition and structure of fungal community are closely related with the function of fine root orders.

[Seasonal dynamics of quantitative and morphological traits of poplar fine roots and their differences between successive rotation plantations].

Based on the fine root samples of the first and second generations of poplar (Populus x euramericana ' Neva'), this study examined the response of quantitative and morphological traits of fine roots of different orders and the difference between generations. The results showed that, the quantitative traits of fine roots, such as root length, root surface area and root biomass, presented obvious seasonal variation, and the fine root traits had obvious difference among root orders. The quantitative traits of lower-order fine roots showed significant seasonal difference, and the fine root biomass increased in the growing season and then decreased significantly. The specific root length (SRL) of higher-order roots also showed significant change with season, while the root length density (RLD) and root tissue density (RTD) changed a little. The successive rotation resulted in the significant increase of root length, root biomass, SRL and RLD of 1-2 orders in the growing season. The quantitative traits of first order root significantly positively correlated with soil temperature and moisture, and significantly negatively correlated with the soil organic matter and soil available nitrogen content. However, the quantitative traits of second order root only showed significant correlation with soil nutrient content. The seasonal dynamics of poplar fine roots and the difference between successive rotation plantations implied carbon investment change of poplar to roots. Soil nutrient deficiency induced more carbon investment into roots, and this carbon allocation pattern might affect the aboveground productivity of poplar plantation.

Transforming growth factor-beta and Wnt signals regulate chondrocyte differentiation through Twist1 in a stage-specific manner.

We investigated the molecular mechanisms underlying the transition between immature and mature chondrocytes downstream of TGF-beta and canonical Wnt signals. We used two developmentally distinct chondrocyte models isolated from the caudal portion of embryonic chick sternum or chick growth plates. Lower sternal chondrocytes exhibited immature phenotypic features, whereas growth plate-extracted cells displayed a hypertrophic phenotype. TGF-beta significantly induced beta-catenin in immature chondrocytes, whereas it repressed it in mature chondrocytes. TGF-beta further enhanced canonical Wnt-mediated transactivation of the Topflash reporter expression in lower sternal chondrocytes. However, it inhibited Topflash activity in a time-dependent manner in growth plate chondrocytes. Our immunoprecipitation experiments showed that TGF-beta induced Sma- and Mad-related protein 3 interaction with T-cell factor 4 in immature chondrocytes, whereas it inhibited this interaction in mature chondrocytes. Similar results were observed by chromatin immunoprecipitation showing that TGF-beta differentially shifts T-cell factor 4 occupancy on the Runx2 promoter in lower sternal chondrocytes vs. growth plate chondrocytes. To further determine the molecular switch between immature and hypertrophic chondrocytes, we assessed the expression and regulation of Twist1 and Runx2 in both cell models upon treatment with TGF-beta and Wnt3a. We show that Runx2 and Twist1 are differentially regulated during chondrocyte maturation. Furthermore, whereas TGF-beta induced Twist1 in mature chondrocytes, it inhibited Runx2 expression in these cells. Opposite effects were observed upon Wnt3a treatment, which predominates over TGF-beta effects on these cells. Finally, overexpression of chick Twist1 in mature chondrocytes dramatically inhibited their hypertrophy. Together, our findings show that Twist1 may be an important regulator of chondrocyte progression toward terminal maturation in response to TGF-beta and canonical Wnt signaling.

Cervical intervertebral disc degeneration induced by unbalanced dynamic and static forces: a novel in vivo rat model.

STUDY DESIGN: Establishment of a novel in vivo animal model of cervical spondylosis. OBJECTIVE: To investigate apoptotic, degenerative, and inflammatory changes occurring in the cervical intervertebral discs of rats. SUMMARY OF BACKGROUND DATA: Cervical degeneration occurs as the result of imbalance of both static and dynamic spinal stabilizers. The disc degeneration that occurs is characterized by increased local inflammation and increased apoptosis of intervertebral disc cells. METHODS: By excising the paraspinal musculature and posterior cervical spinal ligaments of rats, both static and dynamic cervical stabilizers were disrupted. The resultant biomechanical imbalance resulted in biochemical and histologic changes, which were characterized by light microscopy, electron microscopy, immunostaining, enzyme-linked immunosorbent assay, polymerase chain reaction, and in situ hybridization. RESULTS: Histologic analysis showed characteristic degenerative changes of the intervertebral discs and vertebral endplates following surgery. Ultrastructural examination revealed apoptotic changes, which were verified by immunostaining. Instability also resulted in significant up-regulation of inflammatory factors, as shown by enzyme-linked immunosorbent assay, polymerase chain reaction, and in situ hybridization. CONCLUSIONS: By creating static and dynamic posterior instability of the cervical spine, this novel model of cervical spondylosis results in rapid intervertebral disc degeneration characterized by increased apoptosis and local inflammation, such as that seen clinically.

Insulin-like growth factor-1 treatment prevents anti-Fas antibody-induced apoptosis in endplate chondrocytes.

STUDY DESIGN: In vitro investigation of vertebral endplate chondrocyte apoptosis. OBJECTIVES: To determine whether Fas antibody caused apoptosis in endplate chondrocytes, and whether insulin-like growth factor-1 (IGF-1) inhibited this effect. Integrin-alpha1 and focal adhesion kinase (FAK) expression in conjunction with apoptosis was also investigated. SUMMARY OF BACKGROUND DATA: Binding of Fas antibody to Fas mimics Fas-FasL ligation, which causes apoptosis. IGF-1 has been shown to have anti-apoptotic effects. MATERIALS AND METHODS: Rat cervical endplate chondrocytes were cultured and treated with Fas antibody, with or without IGF-1. Cellular morphology was examined by microscopy. Apoptotic changes were evaluated by transmission electron microscopy, TUNEL staining, and immunostaining. Apoptosis-induced changes in the expression of integrin-alpha1 chain and FAK were also investigated. RESULTS: Endplate chondrocytes were able to be cultured; a chondrocytic phenotype was maintained. Fas antibody induced apoptosis in endplate chondrocytes; this was confirmed by TUNEL staining. Bcl-2 expression was decreased by Fas antibody, while Bax expression increased. Integrin-alpha1 and FAK expression was decreased by Fas antibody. IGF-1 treatment inhibited these Fas antibody-induced changes. CONCLUSIONS: Fas antibody induces apoptosis and decreases Integrin-alpha1 and FAK expression in cultured endplate chondrocytes; IGF-1 is protective against these changes.

Wnt induction of chondrocyte hypertrophy through the Runx2 transcription factor.

We investigated the molecular mechanisms underlying canonical Wnt-mediated regulation of chondrocyte hypertrophy using chick upper sternal chondrocytes. Replication competent avian sarcoma (RCAS) viral over-expression of Wnt8c and Wnt9a, upregulated type X collagen (col10a1) and Runx2 mRNA expression thereby inducing chondrocyte hypertrophy. Wnt8c and Wnt9a strongly inhibited mRNA levels of Sox9 and type II collagen (col2a1). Wnt8c further enhanced canonical bone morphogenetic proteins (BMP-2)-induced expression of Runx2 and col10a1 while Wnt8c and Wnt9a inhibited TGF-beta-induced expression of Sox9 and col2a1. Over-expression of beta-catenin mimics the effect of Wnt8c and Wnt9a by upregulating Runx2, col10a1, and alkaline phosphatase (AP) mRNA levels while it inhibits col2a1 transcription. Western blot analysis shows that Wnt8c and beta-catenin also induces Runx2 protein levels in chondrocytes. Thus, our results indicate that activation of the canonical beta-catenin Wnt signaling pathway induces chondrocyte hypertrophy and maturation. We further investigated the effects of beta-catenin-TCF/Lef on Runx2 promoter. Co-transfection of lymphoid enhancer factor (Lef1) and beta-catenin in chicken upper sternal chondrocytes together with deletion constructs of the Runx2 promoter shows that the proximal region spanning the first 128 base pairs of this promoter is responsible for the Wnt-mediated induction of Runx2. Mutation of the TCF/Lef binding site in the -128 fragment of the Runx2 promoter resulted in loss of its responsiveness to beta-catenin. Additionally, gel-shift assay analyses determined the DNA/protein interaction of the TCF/Lef binding sites on the Runx2 promoter. Finally, our site-directed mutagenesis data demonstrated that the Runx2 site on type X collagen promoter is required for canonical Wnt induction of col10a1. Altogether we demonstrate that Wnt/beta-catenin signaling is regulated by TGF-beta and BMP-2 in chick upper sternal chondrocytes, and mediates chondrocyte hypertrophy at least partly through activation of Runx2 which in turn may induce col10a1 expression.

Runx1/AML1/Cbfa2 mediates onset of mesenchymal cell differentiation toward chondrogenesis.

UNLABELLED: Runx proteins mediate skeletal development. We studied the regulation of Runx1 during chondrocyte differentiation by real-time RT-PCR and its function during chondrogenesis using overexpression and RNA interference. Runx1 induces mesenchymal stem cell commitment to the early stages of chondrogenesis. INTRODUCTION: Runx1 and Runx2 are co-expressed in limb bud cell condensations that undergo both cartilage and bone differentiation during murine development. However, the cooperative and/or compensatory effects these factors exert on skeletal formation have yet to be elucidated. MATERIALS AND METHODS: Runx1/Cbfa2 and Runx2/Cbfa1 were examined at different stages of embryonic development by immunohistochemistry. In vitro studies used mouse embryonic limb bud cells and assessed Runx expressions by immunohistochemistry and real-time RT-PCR in the presence and absence of TGFbeta and BMP2. Runx1 was overexpressed in mesenchymal cell progenitors using retroviral infection. RESULTS: Immunohistochemistry showed that Runx1 and Runx2 are co-expressed in undifferentiated mesenchyme, had similar levels in chondrocytes undergoing transition from proliferation to hypertrophy, and that there was primarily Runx2 expression in hypertrophic chondrocytes. Overall, the expression of Runx1 remained significantly higher than Runx2 mRNA levels during early limb bud cell maturation. Treatment of limb bud micromass cultures with BMP2 resulted in early induction of both Runx1 and Runx2. However, upregulation of Runx2 by BMP2 was sustained, whereas Runx1 decreased in later time-points when type X collagen was induced. Although TGFbeta potently inhibits Runx2 and type X collagen, it induces type II collagen mRNA and mildly but significantly inhibits Runx1 isoforms in the early stages of chondrogenesis. Virus-mediated overexpression of Runx1 in mouse embryonic mesenchymal cells resulted in a potent induction of the early chondrocyte differentiation markers but not the hypertrophy marker, type X collagen. Knockdown or Runx1 potently inhibits type II collagen, alkaline phosphatase, and Runx2 and has a late inhibitory effect on type X collagen. CONCLUSION: These findings show a distinct and sustained role for Runx proteins in chondrogenesis and subsequent chondrocyte maturation. Runx1 is highly expressed during chondrogenesis in comparison with Runx2, and Runx1 gain of functions stimulated this process. Thus, the Runx genes are uniquely expressed and have distinct roles during skeletal development.