PdeERF114 recruits PdeWRKY75 to regulate callus formation in poplar by modulating the accumulation of H2 O2 and the relaxation of cell walls.

Callus formation is important for numerous biological processes in plants. Previously, we revealed that the PdeWRKY75-PdeRBOHB module positively regulates hydrogen peroxide (H2 O2 ) accumulation, thereby affecting callus formation in poplar. In this study, we identified and confirmed a transcription factor, PdeERF114, that interacts with PdeWRKY75 both in vitro and in vivo. Gene expression analysis identified both PdeRBOHB and PdeEXPB2 as downstream genes of PdeERF114 and PdeWRKY75. Overexpression (OE) and reduced-expression (RE) transgenic poplar lines for these four genes were generated, and the observation of callus formation was also performed in all plant materials. We demonstrated that PdeERF114 and PdeWRKY75 formed a protein complex and that this complex could bind W-Box motifs in the promoters of PdeRBOHB and PdeEXPB2, thereby positively regulating the expression of PdeRBOHB and PdeEXPB2. The OE/RE transgenic lines for these four genes also showed enhanced/reduced callus formation. Overall, we revealed a novel gene regulatory network for the regulation of callus formation in plants that involves four genes and regulates callus formation through two pathways: the accumulation of H2 O2 in explants and the relaxation of cell walls. In the future, the four genes could be used to enhance transformation effectiveness in genetic engineering.

Gain-of-function of the 1-aminocyclopropane-1-carboxylate synthase gene ACS1G induces female flower development in cucumber gynoecy.

Unisexual flowers provide a useful system for studying plant sex determination. In cucumber (Cucumis sativus L.), three major Mendelian loci control unisexual flower development, Female (F), androecious [a; 1-aminocyclopropane-1-carboxylate {ACC} synthase 11, acs11], and Monoecious (M; ACS2), referred to here as the Female, Androecious, Monoecious (FAM) model, in combination with two genes, gynoecious (g, the WIP family C2H2 zinc finger transcription factor gene WIP1) and the ethylene biosynthetic gene ACC oxidase 2 (ACO2). The F locus, conferring gynoecy and the potential for increasing fruit yield, is defined by a 30.2-kb tandem duplication containing three genes. However, the gene that determines the Female phenotype, and its mechanism, remains unknown. Here, we created a set of mutants and revealed that ACS1G is responsible for gynoecy conferred by the F locus. The duplication resulted in ACS1G acquiring a new promoter and expression pattern; in plants carrying the F locus duplication, ACS1G is expressed early in floral bud development, where it functions with ACO2 to generate an ethylene burst. The resulting ethylene represses WIP1 and activates ACS2 to initiate gynoecy. This early ACS1G expression bypasses the need for ACS11 to produce ethylene, thereby establishing a dominant pathway for female floral development. Based on these findings, we propose a model for how these ethylene biosynthesis genes cooperate to control unisexual flower development in cucumber.

Phosphate (Pi) stress-responsive transcription factors PdeWRKY6 and PdeWRKY65 regulate the expression of PdePHT1;9 to modulate tissue Pi concentration in poplar.

Phosphorus (P) is an important nutrient for plants. Here, we identify a WRKY transcription factor (TF) in poplar (Populus deltoides × Populus euramericana) (PdeWRKY65) that modulates tissue phosphate (Pi) concentrations in poplar. PdeWRKY65 overexpression (OE) transgenic lines showed reduced shoot Pi concentrations under both low and normal Pi availabilities, while PdeWRKY65 reduced expression (RE) lines showed the opposite phenotype. A gene encoding a Pi transporter (PHT), PdePHT1;9, was identified as the direct downstream target of PdeWRKY65 by RNA sequencing (RNA-Seq). The negative regulation of PdePHT1;9 expression by PdeWRKY65 was confirmed by DNA-protein interaction assays, including yeast one-hybrid (Y1H), electrophoretic mobility shift assay (EMSA), co-expression of the promoters of PdePHT1;9 and PdeWRKY65 in tobacco (Nicotiana benthamiana) leaves, and chromatin immunoprecipitation-quantitative PCR. A second WRKY TF, PdeWRKY6, was subsequently identified and confirmed to positively regulate the expression of PdePHT1;9 by DNA-protein interaction assays. PdePHT1;9 and PdeWRKY6 OE and RE poplar transgenic lines were used to confirm their positive regulation of shoot Pi concentrations, under both normal and low Pi availabilities. No interaction between PdeWRKY6 and PdeWRKY65 was observed at the DNA or protein levels. Collectively, these data suggest that the low Pi-responsive TFs PdeWRKY6 and PdeWRKY65 independently regulate the expression of PHT1;9 to modulate tissue Pi concentrations in poplar.

The transcription factor WRKY75 regulates the development of adventitious roots, lateral buds and callus by modulating hydrogen peroxide content in poplar.

Hydrogen peroxide (H2O2) plays important roles in plant development. Adventitious roots (AR), lateral buds (LB) and callus formation are important traits for plants. Here, a gene encoding RESPIRATORY BURST OXIDASE HOMOLOG B (PdeRBOHB) from poplar line 'NL895' (Populus. deltoides × P. euramericana) was predicted to be involved in H2O2 accumulation, and lines with reduced expression were generated. H2O2 content was decreased, and the development of adventitious roots, lateral buds, and callus was inhibited in reduced expression PdeRBOHB lines. A gene encoding PdeWRKY75 was identified as the upstream transcription factor positively regulating PdeRBOHB. This regulation was confirmed by dual luciferase reporter assay, GUS transient expression analysis and electrophoretic mobility shift assay. In the reduced expression PdeWRKY75 lines, H2O2 content was decreased and the development of adventitious roots, lateral buds, and callus development was inhibited, while in the overexpression lines, H2O2 content was increased and the development of adventitious roots and lateral buds was inhibited, but callus formation was enhanced. Additionally, reduced expression PdeRBOHB lines showed lowered expression of PdeWRKY75, while exogenous application of H2O2 showed the opposite effect. Together, these results suggest that PdeWRKY75 and PdeRBOHB are part of a regulatory module in H2O2 accumulation, which is involved in the regulation of multiple biological processes.

Sewage sludge ash-incorporated stabilisation/solidification for recycling and remediation of marine sediments.

Finding suitable disposal sites for dredged marine sediments and incinerated sewage sludge ash (ISSA) is a challenge. Stabilisation/solidification (S/S) has become an increasingly popular remediation technology. This study sheds light on the possible beneficial use of ISSA together with traditional binders to stabilise/solidify marine sediments. The performance of the binders on S/S of sediment 1 (clean) and sediment 2 (contaminated) was also compared. The results showed that the use of ISSA as part of the binder was effective in promoting the strength of the sediment with a high initial moisture content due to ISSA porous and high water absorption characteristics. The sediments treated with 10% cement and 20% ISSA attained the highest strength. Also, cement hydration as well as pozzolanic reactions between ISSA and Ca(OH)2 made contributions to the strength development. This was supported by the microstructural analysis, in particular the porosity results. In terms of environmental impacts, two leaching tests (toxicity characteristic leaching procedure and synthetic precipitation leaching procedure) found that all the S/S treated sediment by 10% lime and 20% ISSA resulted in the lowest leachate concentrations under the on-site reuse scenario or under simulative acidic rainfall conditions. Therefore, recycling waste ISSA with lime can be used as an appealing binder to replace cement to stabilise/solidify dredged marine sediments for producing fill materials.

Uncovering the Role of PdePrx12 Peroxidase in Enhancing Disease Resistance in Poplar Trees.

Peroxidase (Prx)-related genes are reported to be involved in the metabolism of hydrogen peroxide (H2O2) in plants. Here, we found that the expression of the PdePrx12 gene was upregulated in wild-type (WT) poplar line NL895 infected with the pathogens Botryosphaeria dothidea strain 3C and Alternaria alternata strain 3E. The PdePrx12 gene was cloned in the poplar line NL895 and its overexpression (OE) and reduced-expression (RE) vectors were constructed. OE and RE transgenic lines were then generated. The H2O2 content in the leaves was measured by DAB staining and spectrophotometric analysis, and the data revealed that the OE line had a reduced H2O2 content, whereas the RE line had an increased H2O2 content. These transgenic and WT plants were also inoculated with the 3C/3E pathogens. The leaf area infected by pathogen 3C/3E was determined and the OE line was found to have a larger area of infection, whereas the RE line was found to have a smaller area of infection. This result suggested PdePRX12 is involved in disease resistance in poplar. Given these results, this study demonstrated that when poplar is infected by pathogens, the expression of PdePrx12 is inhibited, leading to an increase in H2O2 content, thereby enhancing disease resistance.

Isolation, identification and bioactivity of endophytic fungi from medicinal plant Malus sieboldii.

OBJECTIVE: To isolate and identify endophytic fungi from Malus sieboldii, and detect cytotoxicity, protease inhibition and antifungal activities of their crude extracts. METHOD: The fungi were identified with the aid of morphology or Internal Transcribed Spacer (ITS) rDNA molecular methods. Fungal activities were tested by cylinder-plate, MTT and BRpNA methods, respectively. RESULT: A total of 217 endophytic fungi were isolated from M. sieboldii. Of the 22 taxa obtained, non-sporulating, Alternaria, Colletotrichum, Aspergillu, Fusarlum, Gliocladium and Cunninghamella were dominant communities. The result of the bioactivity test showed that 30 endophytic fungi displayed inhibition against at least one pathogenic fungus, and 3 and 4 showed cytotoxicity and protease inhibition, respectively. CONCLUSION: M. sieboldii should be a potential source of bioactive endophytic fungi.

Experimental Study on Carbonation of Cement-Based Materials in Underground Engineering.

The corrosive water environment has a decisive influence on the durability of a diversion tunnel lining. In this paper, the effects of carbonation on cement-based materials in water-immersion and saturated-humidity environments were studied by increasing the CO2 concentration. The results show that under conditions of water-immersion and saturated humidity, the color of the non-carbonation region is dark, while the carbonation region is gray, and the color boundary is obvious. However, in an atmospheric environment, there is no zone with a dark color and the color boundary is not obvious. In a saturated-humidity environment, the carbonation depth increases over time and changes greatly, and its value is about 16.71 mm at 200 days. While in a water-immersion environment, the carbonation depth varies little with time and the value is only 2.31 mm. The carbonation depths of cement mortar samples in different environments generally follow a linear relationship with the square root of time. The carbonation coefficient in a saturated-humidity environment is more than nine times that in the water-immersion environment. In a water-immersion environment, the carbonation causes a large loss of calcium in cement-based materials, and their Ca/Si ratio obviously decreases. The calcium silicon ratio (Ca/Si) of cement-based materials in a water-immersion environment is 0.11, which is much less than 1.51 in a water-saturated environment and 1.49 in an atmospheric environment. In a saturated-humidity environment, the carbonation only reduces the pH of the pore solution in the carbonation region, and the structural stability of cement-based materials is not degraded. The number of pores of all radii after carbonation in a water-immersion environment exceeds that in a saturated-humidity environment, and the total pore volume and average pore radius in a water-immersion environment are also larger than in a saturated-humidity environment, so the water-immersion environment accelerates the development and expansion of pores. The research results can provide some theoretical and technical support for the design, construction, and safe operation of diversion tunnel linings.

Investigation on Water Transformation and Pore Structure of Cement-Stabilized Dredged Sediment Based on NMR Technology.

Cement-stabilized dredged sediment (CDS) when used as a new road construction material cannot only solve the problem of abandoned sediment disposal, but also effectively save natural soil resources. This study aimed to evaluate the strength and permeability of CDS and establish corresponding prediction models from the perspective of a stabilization mechanism. The soil-water composition and pore size distribution were investigated by the nuclear magnetic resonance (NMR) technique. The results demonstrated that more liquid pore water inside the CDS specimen transformed into combined water with cement hydration. The amount of combined water, which essentially characterized the hydration process of cement, presented a linear relationship with log (t). The cementation and filling action of hydrates resulted in the transformation of large pores into smaller ones, hence the optimal pore size decreased with an increasing curing period and cement content. The stress-strain curves and hydraulic conductivity were determined based on unconfined compression and flexible wall penetration tests, respectively. The unconfined compressive strength increased exponentially with the amount of combined water, and the functional correlations of hydraulic conductivity and micropore parameters were established. The reliability of the NMR technique as a new method to study the microscopic evolution mechanism of the strength and permeability of CDS was further verified by scanning electron microscopy and mercury intrusion porosimetry tests.

The chromosome-scale genome sequence of Triadica sebifera provides insight into fatty acids and anthocyanin biosynthesis.

The Chinese tallow tree (Triadica sebifera) can produce oil with high content of unsaturated fatty acids in seeds and shows attractive leaf color in autumn and winter. Here, the 739 Mb chromosome-scale genome sequence of the Chinese tallow tree was assembled and it reveals the Chinese tallow tree is a tetraploid. Numerous genes related to nutrition assimilation, energy utilization, biosynthesis of secondary metabolites and resistance significantly expanded or are specific to the Chinese tallow tree. These genes would enable the Chinese tallow tree to obtain high adaptability. More genes in fatty acids biosynthesis in its genome, especially for unsaturated fatty acids biosynthesis, and higher expression of these genes in seeds would be attributed to its high content of unsaturated fatty acids. Cyanidin 3-O-glucoside was identified as the major component of anthocyanin in red leaves. All structural genes in anthocyanin biosynthesis show significantly higher expression in red leaves than in green leaves. Transcription factors, seven MYB and one bHLH, were predicted to regulate these anthocyanin biosynthesis genes. Collectively, we provided insight into the polyploidization, high adaptability and biosynthesis of the high content of unsaturated fatty acids in seeds and anthocyanin in leaves for the Chinese tallow tree.