A COBRA family protein, PtrCOB3, contributes to gelatinous layer formation of tension wood fibers in poplar.

Angiosperm trees usually develop tension wood (TW) in response to gravitational stimulation. TW comprises abundant gelatinous (G-) fibers with thick G-layers primarily composed of crystalline cellulose. Understanding of the pivotal factors governing G-layer formation in TW fiber remains elusive. This study elucidates the role of a Populus trichocarpa COBRA family protein, PtrCOB3, in the G-layer formation of TW fibers. PtrCOB3 expression was upregulated, and its promoter activity was enhanced during TW formation. Comparative analysis with wild-type trees revealed that ptrcob3 mutants, mediated by Cas9/gRNA gene editing, were incapable of producing G-layers within TW fibers and showed severely impaired stem lift. Fluorescence immunolabelling data revealed a dearth of crystalline cellulose in the tertiary cell wall (TCW) of ptrcob3 TW fibers. The role of PtrCOB3 in G-layer formation is contingent upon its native promoter, as evidenced by the comparative phenotypic assessments of pCOB11::PtrCOB3, pCOB3::PtrCOB3, and pCOB3::PtrCOB11 transgenic lines in the ptrcob3 background. Overexpression of PtrCOB3 under the control of its native promoter expedited G-layer formation within TW fibers. We further identified three transcription factors that bind to the PtrCOB3 promoter and positively regulate its transcriptional levels. Alongside the primary TCW synthesis genes, these findings enable the construction of a two-layer transcriptional regulatory network for the G-layer formation of TW fibers. Overall, this study uncovers mechanistic insight into TW formation, whereby a specific COB protein executes the deposition of cellulose, and consequently, G-layer formation within TW fibers.

Genome-Wide Identification of TLP Gene Family in Populus trichocarpa and Functional Characterization of PtTLP6, Preferentially Expressed in Phloem.

Thaumatin-like proteins (TLPs) in plants are involved in diverse biotic and abiotic stresses, including antifungal activity, low temperature, drought, and high salinity. However, the roles of the TLP genes are rarely reported in early flowering. Here, the TLP gene family was identified in P. trichocarpa. The 49 PtTLP genes were classified into 10 clusters, and gene structures, conserved motifs, and expression patterns were analyzed in these PtTLP genes. Among 49 PtTLP genes, the PtTLP6 transcription level is preferentially high in stems, and GUS staining signals were mainly detected in the phloem tissues of the PtTLP6pro::GUS transgenic poplars. We generated transgenic Arabidopsis plants overexpressing the PtTLP6 gene, and its overexpression lines showed early flowering phenotypes. However, the expression levels of main flowering regulating genes were not significantly altered in these PtTLP6-overexpressing plants. Our data further showed that overexpression of the PtTLP6 gene led to a reactive oxygen species (ROS) burst in Arabidopsis, which might advance the development process of transgenic plants. In addition, subcellular localization of PtTLP6-fused green fluorescent protein (GFP) was in peroxisome, as suggested by tobacco leaf transient transformation. Overall, this work provides a comprehensive analysis of the TLP gene family in Populus and an insight into the role of TLPs in woody plants.

Time transfer over 113†km free space laser communication channel.

The space time frequency transfer plays a crucial role in applications such as space optical clock networks, navigation, satellite ranging, and space quantum communication. Here, we propose a high-precision space time frequency transfer and time synchronization scheme based on a simple intensity modulation/direct detection (IM/DD) laser communication system, which occupies a communication bandwidth of approximately 0.2%. Furthermore, utilizing an optical-frequency comb time frequency transfer system as an out-of-loop reference, experimental verification was conducted on a 113 km horizontal atmospheric link, with a long-term stability approximately 8.3 × 10-16 over a duration of 7800 seconds. Over an 11-hour period, the peak-to-peak wander is approximately 100 ps. Our work establishes the foundation of the time frequency transfer, based on the space laser communication channel, for future ground-to-space and inter-satellite links.

Spatiotemporal analysis of national carbon emission and regional carbon simulation in China.

Land use and land cover (LULC) will cause large flows of carbon sources and sinks. As the world's largest carbon emitter with a complicated LULC, China's carbon emissions have profound implications for its ecological environment and future development. In this paper, we account for the land-use changes and carbon emissions of 30 Chinese provinces and cities in China from 2000 to 2020. Furthermore, the spatial correlation of carbon emissions among the study areas is explored. Four typical regions with spatial association (Beijing, Hebei, Sichuan, and Anhui) are selected, and their land-use change trends in 2025 and 2030 are simulated to predict the total carbon emissions in the future. The results show that the distribution of land-use in China is mainly cultivated and woodland, but the growth of urban built-up (UBL) land area indirectly leads to the continuous increase of carbon emissions. Total carbon emissions have increased over the past two decades, albeit at a slower growth rate, with some provinces experiencing no further growth. In the typical regional carbon emission simulation, it is found that the carbon emissions of the four provinces would show a downward trend in the future. The main reason is the reduction in indirect carbon emissions from fossil energy in UBL, while the other part is the influx of carbon sinks due to grassland, woodland, etc. We recommended that future carbon reduction measures should focus and prioritize controlling fossil energy and mitigating carbon emissions from UBL. Simultaneously, the significant contribution of forests and other land types as carbon sinks should be acknowledged to better implement China's carbon neutral commitment.

Arabidopsis pollen-specific glycerophosphodiester phosphodiesterase-like genes are essential for pollen tube tip growth.

In angiosperms, pollen tube growth is critical for double fertilization and seed formation. Many of the factors involved in pollen tube tip growth are unknown. Here, we report the roles of pollen-specific GLYCEROPHOSPHODIESTER PHOSPHODIESTERASE-LIKE (GDPD-LIKE) genes in pollen tube tip growth. Arabidopsis thaliana GDPD-LIKE6 (AtGDPDL6) and AtGDPDL7 were specifically expressed in mature pollen grains and pollen tubes and green fluorescent protein (GFP)-AtGDPDL6 and GFP-AtGDPDL7 fusion proteins were enriched at the plasma membrane at the apex of forming pollen tubes. Atgdpdl6 Atgdpdl7 double mutants displayed severe sterility that was rescued by genetic complementation with AtGDPDL6 or AtGDPDL7. This sterility was associated with defective male gametophytic transmission. Atgdpdl6 Atgdpdl7 pollen tubes burst immediately after initiation of pollen germination in vitro and in vivo, consistent with the thin and fragile walls in their tips. Cellulose deposition was greatly reduced along the mutant pollen tube tip walls, and the localization of pollen-specific CELLULOSE SYNTHASE-LIKE D1 (CSLD1) and CSLD4 was impaired to the apex of mutant pollen tubes. A rice pollen-specific GDPD-LIKE protein also contributed to pollen tube tip growth, suggesting that members of this family have conserved functions in angiosperms. Thus, pollen-specific GDPD-LIKEs mediate pollen tube tip growth, possibly by modulating cellulose deposition in pollen tube walls.

Overexpression of histone deacetylase gene 84KHDA909 from poplar confers enhanced tolerance to drought and salt stresses in Arabidopsis.

Histone deacetylases (HDACs) are important enzymes participating in histone modification and epigenetic regulation of gene transcription. HDACs play an essential role in plant development and stress responses. To date, the role of HDACs is largely uninvestigated in woody plants. In this study, we identified a RPD3/HDA1-type HDAC, named 84KHDA909, from 84 K poplar (Populus alba × Populus glandulosa). The protein encoded by 84KHDA909 contained an HDAC domain. The 84KHDA909 was responsive to drought, salt, and cold stresses, but displayed different expression patterns. Overexpression of 84KHDA909 improved root growth, and conferred enhanced tolerance to drought and salt stresses in Arabidopsis. The transgenic plants displayed greater fresh weight, higher proline content and lower malondialdehyde (MDA) accumulation than the wild type. In the transgenic plants, transcript levels of several genes related to abscisic acid (ABA) biosynthesis and response were altered upon exposure to drought and salt stresses. Our results suggested that 84KHDA909 positively regulates drought and salt stress tolerance through ABA pathway.

Aspartic proteases modulate programmed cell death and secondary cell wall synthesis during wood formation in poplar.

Programmed cell death (PCD) is essential for wood development in trees. However, the determination of crucial factors involved in xylem PCD of wood development is still lacking. Here, two Populus trichocarpa typical aspartic protease (AP) genes, AP17 and AP45, modulate xylem maturation, especially fibre PCD, during wood formation. AP17 and AP45 were dominantly expressed in the fibres of secondary xylem, as suggested by GUS expression in APpro::GUS transgenic plants. Cas9/gRNA-induced AP17 or AP45 mutants delayed secondary xylem fibre PCD, and ap17ap45 double mutants showed more serious defects. Conversely, AP17 overexpression caused premature PCD in secondary xylem fibres, indicating a positive modulation in wood fibre PCD. Loss of AP17 and AP45 did not alter wood fibre wall thickness, whereas the ap17ap45 mutants showed a low lignin content in wood. However, AP17 overexpression led to a significant decrease in wood fibre wall thickness and lignin content, revealing the involvement in secondary cell wall synthesis during wood formation. In addition, the ap17ap45 mutant and AP17 overexpression plants resulted in a significant increase in saccharification yield in wood. Overall, AP17 and AP45 are crucial modulators in xylem maturation during wood development, providing potential candidate genes for engineering lignocellulosic wood for biofuel utilization.

A CEEMD-ARIMA-SVM model with structural breaks to forecast the crude oil prices linked with extreme events.

This paper develops an integrated framework to forecast the volatility of crude oil prices by considering the impacts of extreme events (structural breaks). The impacts of extreme events are vital to improving prediction accuracy. Aiming to demonstrate the crude oil price fluctuation and the impacts of external events, this paper employs the complementary ensemble empirical mode decomposition (CEEMD). It decomposes the crude oil price into some constituents at various frequencies to extract a market fluctuation, a shock from extreme events and a long-term trend. The shock from extreme events is found to be the most crucial element in deciding the crude oil prices. Then we combine the iterative cumulative sum of squares (ICSS) test with the Chow test to get the structural breaks and analyze the extreme event impacts. Finally, this paper combines the structural breaks, the autoregressive integrated moving average (ARIMA) model, and the support vector machine (SVM) to make a forecast of the crude oil prices. The empirical process proves that the CEEMD-ARIMA-SVM model with structural breaks performs the best when compared with the other ARIMA-type models and SVM-type models. The framework offers an insightful view to help decision-makers and can be used in many areas. Supplementary Information: The online version contains supplementary material available at 10.1007/s00500-022-07276-5.

Role of lncRNAs in cis- and trans-regulatory responses to salt in Populus trichocarpa.

Long non-coding RNAs (lncRNAs) are emerging as versatile regulators in diverse biological processes. However, little is known about their cis- and trans-regulatory contributions in gene expression under salt stress. Using 27 RNA-seq data sets from Populus trichocarpa leaves, stems and roots, we identified 2988 high-confidence lncRNAs, including 1183 salt-induced differentially expressed lncRNAs. Among them, 301 lncRNAs have potential for positively affecting their neighboring genes, predominantly in a cis-regulatory manner rather than by co-transcription. Additionally, a co-expression network identified six striking salt-associated modules with a total of 5639 genes, including 426 lncRNAs, and in these lncRNA sequences, the DNA/RNA binding motifs are enriched. This suggests that lncRNAs might contribute to distant gene expression of the salt-associated modules in a trans-regulatory manner. Moreover, we found 30 lncRNAs that have potential to simultaneously cis- and trans-regulate salt-responsive homologous genes, and Ptlinc-NAC72, significantly induced under long-term salt stress, was selected for validating its regulation of the expression and functional roles of the homologs PtNAC72.A and PtNAC72.B (PtNAC72.A/B). The transient transformation of Ptlinc-NAC72 and a dual-luciferase assay of Ptlinc-NAC72 and PtNAC72.A/B promoters confirmed that Ptlinc-NAC72 can directly upregulate PtNAC72.A/B expression, and a presence/absence assay was further conducted to show that the regulation is probably mediated by Ptlinc-NAC72 recognizing the tandem elements (GAAAAA) in the PtNAC72.A/B 5' untranslated region (5'-UTR). Finally, the overexpression of Ptlinc-NAC72 produces a hypersensitive phenotype under salt stress. Altogether, our results shed light on the cis- and trans-regulation of gene expression by lncRNAs in Populus and provides an example of long-term salt-induced Ptlinc-NAC72 that could be used to mitigate growth costs by conferring plant resilience to salt stress.

Cas9/gRNA-Mediated Mutations in PtrFLA40 and PtrFLA45 Reveal Redundant Roles in Modulating Wood Cell Size and SCW Synthesis in Poplar.

Fasciclin-like arabinogalactan proteins (FLAs) play an important role in plant development and adaptation to the environment. However, the roles of FLAs in wood formation remain poorly understood. Here, we identified a total of 50 PtrFLA genes in poplar. They were classified into four groups: A to D, among which group A was the largest group with 28 members clustered into four branches. Most PtrFLAs of group A were dominantly expressed in developing xylem based on microarray and RT-qPCR data. The roles of PtrFLA40 and PtrFLA45 in group A were investigated via the Cas9/gRNA-induced mutation lines. Loss of PtrFLA40 and PtrFLA45 increased stem length and diameter in ptrfla40ptrfla45 double mutants, but not in ptrfla40 or ptrfla45 single mutants. Further, our findings indicated that the ptrfla40ptrfla45 mutants enlarged the cell size of xylem fibers and vessels, suggesting a negative modulation in stem xylem cell size. In addition, wood lignin content in the ptrfla40fla45 mutants was increased by nearly 9%, and the lignin biosynthesis-related genes were significantly up-regulated in the ptrfla40fla45 mutants, in agreement with the increase in wood lignin content. Overall, Cas9/gRNA-mediated mutations in PtrFLA40 and PtrFLA45 reveal redundant roles in modulating wood cell size and secondary cell wall (SCW) synthesis in poplar.

Effect of thermophilic bacterium HB27 manganese superoxide dismutase in a rat model of chronic prostatitis/chronic pelvic pain syndrome (CP/CPPS).

We investigated the therapeutic effects of superoxide dismutase (SOD) from thermophilic bacterium HB27 on chronic prostatitis/chronic pelvic pain syndrome (CP/CPPS) and its underlying mechanisms. A Sprague-Dawley rat model of CP/CPPS was prepared and then administered saline or Thermus thermophilic (Tt)-SOD intragastrically for 4 weeks. Prostate inflammation and fibrosis were analyzed by hematoxylin and eosin staining, and Masson staining. Alanine transaminase (ALT), aspartate transaminase (AST), serum creatinine (CR), and blood urea nitrogen (BUN) levels were assayed for all animals. Enzyme-linked immunosorbent assays (ELISA) were performed to analyze serum cytokine concentrations and tissue levels of malondialdehyde, nitric oxide, SOD, catalase, and glutathione peroxidase. Reactive oxygen species levels were detected using dichlorofluorescein diacetate. The messenger ribonucleic acid (mRNA) expression of tissue cytokines was analyzed by reverse transcription polymerase chain reaction (RT-PCR), and infiltrating inflammatory cells were examined using immunohistochemistry. Nuclear factor-κB (NF-κB) P65, P38, and inhibitor of nuclear factor-κBα (I-κBα) protein levels were determined using western blot. Tt-SOD significantly improved histopathological changes in CP/CPPS, reduced inflammatory cell infiltration and fibrosis, increased pain threshold, and reduced the prostate index. Tt-SOD treatment showed no significant effect on ALT, AST, CR, or BUN levels. Furthermore, Tt-SOD reduced inflammatory cytokine expression in prostate tissue and increased antioxidant capacity. This anti-inflammatory activity correlated with decreases in the abundance of cluster of differentiation 3 (CD3), cluster of differentiation 45 (CD45), and macrophage inflammatory protein 1α (MIP1α) cells. Tt-SOD alleviated inflammation and oxidative stress by reducing NF-κB P65 and P38 protein levels and increasing I-κBα protein levels. These findings support Tt-SOD as a potential drug for CP/CPPS.

Functional understanding of secondary cell wall cellulose synthases in Populus trichocarpa via the Cas9/gRNA-induced gene knockouts.

Plant cellulose is synthesized by a large plasma membrane-localized cellulose synthase (CesA) complex. However, an overall functional determination of secondary cell wall (SCW) CesAs is still lacking in trees, especially one based on gene knockouts. Here, the Cas9/gRNA-induced knockouts of PtrCesA4, 7A, 7B, 8A and 8B genes were produced in Populus trichocarpa. Based on anatomical, immunohistochemical and wood composition evidence, we gained a comprehensive understanding of five SCW PtrCesAs at the genetic level. Complete loss of PtrCesA4, 7A/B or 8A/B led to similar morphological abnormalities, indicating similar and nonredundant genetic functions. The absence of the gelatinous (G) layer, one-layer-walled fibres and a 90% decrease in cellulose in these mutant woods revealed that the three classes of SCW PtrCesAs are essential for multilayered SCW structure and wood G-fibre. In addition, the mutant primary and secondary phloem fibres lost the n(G + L)- and G-layers and retained the thicker S-layers (L, lignified; S, secondary). Together with polysaccharide immunolocalization data, these findings suggest differences in the role of SCW PtrCesAs-synthesized cellulose in wood and phloem fibre wall structures. Overall, this functional understanding of the SCW PtrCesAs provides further insights into the impact of lacking cellulose biosynthesis on growth, SCW, wood G-fibre and phloem fibre wall structures in the tree.

Navigation Grade MEMS IMU for A Satellite.

This paper presents a navigation grade micro-electromechanical system (MEMS) inertial measurement unit (IMU) that was successfully applied for the first time in the Lobster-Eye X-ray Satellite in July 2020. A six-axis MEMS gyroscope redundant configuration is adopted in the unit to improve the performance through mutual calibration of a set of two-axis gyroscopes in the same direction. In the paper, a satisfactory precision of the gyroscope is achieved by customized and self-calibration gyroscopes whose parameters are adjusted at the expense of bandwidth and dynamics. According to the in-orbit measured data, the MEMS IMU provides an outstanding precision of better than 0.02 °/h (1σ) with excellent bias instability of 0.006 °/h and angle random walk (ARW) of around 0.003 °/h1/2. It is the highest precision MEMS IMU for commercial aerospace use ever publicly reported in the world to date.

Analysis of the energy source at the early stage of poplar seed germination: verification of Perl's pathway.

Adenosine triphosphate (ATP) is produced at the early stage of seed germination and provides the energy for metabolism. The source of ATP in seeds may be Perl's pathway, but this has not yet been confirmed. In this study, using germinating seeds of poplar as the experimental materials, the transcript levels of genes related to Perl's pathway were determined by real-time PCR. The activities of enzymes in Perl's pathway were also determined. The results were verified by comparison with RNA-Seq and metabolomics data. The results showed that there were high transcript levels of some genes encoding malate dehydrogenase (MDH), phosphoenolpyruvate carboxykinase (PEPCK), pyruvate decarboxylase (PDC), alcohol dehydrogenase (ADH), and pyruvate kinase (PK) at the early stage of germination (0.75 h). The enzymes MDH, PEPCK, PK, PDC, and ADH showed peaks in activity at around 0.75 h and 6 h during germination. The oxaloacetate concentration was high in poplar seeds at the early stage of germination. This study provides experimental data showing that Perl's pathway participates in supplying energy during the early stages of poplar seed germination, and lays the foundation for further studies on the complex metabolic processes that function during seed germination.

A systematic approach of determining compensation and allocation for river basin water environment based on total pollutants control.

The compensation of upstream protective area should be determined carefully. This paper provides a comprehensive evaluation framework for economic assessments on the treatment of wastewater discharged into a river basin. We explore the rational costs of compensation and the funding allocation in the Xin'an River basin based on rate agreements. The compensation is determined in accordance with the total compensation model based on the opportunity cost method and allocated by the method of information entropy. In this study, we identify the total compensation payment and distribution of funds in each district and county in the upstream area (Huangshan). Results show that the She county can receive the highest compensation, and the Tunxi district deserves the lowest compensation. In addition, this paper demonstrates that the existing compensation is insufficient for the reduction of water pollutants in the upstream area. Our findings contribute to the existing schemes of ecosystem services payment and improve the environmental decision-making.

Hydraulic Fracturing Experiment Investigation for the Application of Geothermal Energy Extraction.

As an attractive renewable energy source, deep geothermal energy is increasingly explored. Granite is a typical geothermal reservoir rock type with low permeability, and hydraulic fracturing is a promising reservoir stimulation method which could obviously enhance the reservoir permeability. Previous hydraulic fracturing studies were mostly conducted on artificial samples and small cylindrical granites. The fracturing pressures of artificial samples and small real rock sample were much lower than that of field operation, and it was difficult to observe morphological changes in small rocks. Hence, this paper presents a hydraulic fracturing experimental study on large-scale granite with a sample size of 300 × 300 × 300 mm under high temperatures. Besides, injection flow rate is an important parameter for on-site hydraulic fracturing; previous studies usually only focused on breakdown pressure, and there is a lack of comprehensive analysis about fracturing pressure curves and fracturing characteristics caused by different injection flow rates. This study aims to investigate the influence of injection flow rate on different pressure curve characteristic parameters which are initiation pressure, propagation time, breakdown pressure, postfracturing pressure, fracture geometry, and fracture permeability. The mean injection power was proposed to roughly estimate the fracture total lengths. These results could provide some guidance for field-scale reservoir stimulation and heat extraction efficiency improvement.

Histone deacetylase gene PtHDT902 modifies adventitious root formation and negatively regulates salt stress tolerance in poplar.

Histone deacetylases (HDACs) regulate gene transcription, and play a critical role in plant growth, development and stress responses. HD2 proteins are plant specific histone deacetylases. In woody plants, functions of HD2s are not known. In this study, we cloned an HD2 gene PtHDT902 from Populus trichocarpa and investigated its sequence, expression, subcellular localization, and functions in root development and salt stress responses. Our findings indicated that PtHDT902 was a nuclear protein and its expression was regulated by abiotic stresses. The over-expression of PtHDT902 in both Arabidopsis and poplar increased the expression levels of gibberellin (GA) biosynthetic genes. The expression of PtHDT902 in Arabidopsis enhanced primary root growth, and its over-expression in poplar inhibited adventitious root formation. These phenotypes resulted from over-expression of PtHDT902 were consistent with the GA-overproduction phenotypes. In addition, the poplar plants over-expressing PtHDT902 exhibited lower tolerance to salt than non-transgenic plants. These findings indicated that PtHDT902 worked as an important regulator in adventitious root formation and salt stress tolerance in poplar.

Genome-wide characterization of aspartic protease (AP) gene family in Populus trichocarpa and identification of the potential PtAPs involved in wood formation.

BACKGROUND: Aspartic protease (AP) is one of four large proteolytic enzyme families that are involved in plant growth and development. Little is known about the AP gene family in tree species, although it has been characterized in Arabidopsis, rice and grape. The AP genes that are involved in tree wood formation remain to be determined. RESULTS: A total of 67 AP genes were identified in Populus trichocarpa (PtAP) and classified into three categories (A, B and C). Chromosome mapping analysis revealed that two-thirds of the PtAP genes were located in genome duplication blocks, indicating the expansion of the AP family by segmental duplications in Populus. The microarray data from the Populus eFP browser demonstrated that PtAP genes had diversified tissue expression patterns. Semi-qRT-PCR analysis further determined that more than 10 PtAPs were highly or preferentially expressed in the developing xylem. When the involvement of the PtAPs in wood formation became the focus, many SCW-related cis-elements were found in the promoters of these PtAPs. Based on PtAPpromoter::GUS techniques, the activities of PtAP66 promoters were observed only in fiber cells, not in the vessels of stems as the xylem and leaf veins developed in the transgenic Populus tree, and strong GUS signals were detected in interfascicular fiber cells, roots, anthers and sepals of PtAP17promoter::GUS transgenic plants. Intensive GUS activities in various secondary tissues implied that PtAP66 and PtAP17 could function in wood formation. In addition, most of the PtAP proteins were predicted to contain N- and (or) O-glycosylation sites, and the integration of PNGase F digestion and western blotting revealed that the PtAP17 and PtAP66 proteins were N-glycosylated in Populus. CONCLUSIONS: Comprehensive characterization of the PtAP genes suggests their functional diversity during Populus growth and development. Our findings provide an overall understanding of the AP gene family in trees and establish a better foundation to further describe the roles of PtAPs in wood formation.

Simple, rapid and efficient transformation of genotype Nisqually-1: a basic tool for the first sequenced model tree.

Genotype Nisqually-1 is the first model woody plant with an available well-annotated genome. Nevertheless, a simple and rapid transformation of Nisqually-1 remains to be established. Here, we developed a novel shoot regeneration method for Nisqually-1 using leaf petiole and stem segment explants. Numerous shoots formed in the incision of explants within two weeks. The optimized shoot regeneration medium (SRM) contained 0.03 mg l-1 6-benzylaminopurine, 0.02 mg l-1 indole-3-butyric acid and 0.0008 mg l-1 thidiazuron. Based on this, Agrobacterium-mediated genetic transformation of stem explants was examined using the vector pBI121 that contains the ß-glucuronidase (GUS) as a reporter gene. Consequently, factors affecting transformation frequency of GUS-positive shoots were optimized as follows: Agrobacteria cell suspension with an OD600 of 0.4, 20 min infection time, 2 days of co-cultivation duration and the addition of 80 µM acetosyringone into Agrobacteria infective suspension and co-cultivation SRM. Using this optimized method, transgenic plantlets of Nisqually-1 - with an average transformation frequency of 26.7% - were obtained with 2 months. Southern blot and GUS activity staining confirmed the integration of the foreign GUS gene into Nisqually-1. This novel transformation system for Nisqually-1 was rapid, efficient, and simple to operate and will improve more genetic applications in this model tree.

Functional Characterization of Populus PsnSHN2 in Coordinated Regulation of Secondary Wall Components in Tobacco.

Wood formation is a biological process during which the most abundant lignocellulosic biomass on earth is produced. Although a number of transcription factors have been linked to the regulation of wood formation process, none of them has been demonstrated to be a higher hierarchical regulator that coordinately regulates secondary wall biosynthesis genes. Here, we identified a Populus gene, PsnSHN2, a counterpart of the Arabidopsis AP2/ERF type transcription factor, SHINE2. PsnSHN2 is predominantly expressed in xylem tissues and acted evidently as a high hierarchical transcriptional activator. Overexpression of PsnSHN2 in tobacco significantly altered the expression of both transcription factors and biosynthesis genes involved in secondary wall formation, leading to the thickened secondary walls and the changed cell wall composition. The most significant changes occurred in the contents of cellulose and hemicellulose that increased 37% and 28%, respectively, whereas the content of lignin that decreased 34%. Furthermore, PsnSHN2 activated or repressed the promoter activities of transcription factors involved in secondary wall biosynthesis and bound to five cis-acting elements enriched in the promoter regions of these transcription factors. Taken together, our results suggest PsnSHN2 coordinately regulate secondary wall formation through selective up/down-regulation of its downstream transcription factors that control secondary wall formation.