Maize auxin response factor ZmARF1 confers multiple abiotic stresses resistances in transgenic Arabidopsis.

Prolonged exposure to abiotic stresses causes oxidative stress, which affects plant development and survival. In this research, the overexpression of ZmARF1 improved tolerance to low Pi, drought and salinity stresses. The transgenic plants manifested tolerance to low Pi by their superior root phenotypic traits: root length, root tips, root surface area, and root volume, compared to wide-type (WT) plants. Moreover, the transgenic plants exhibited higher root and leaf Pi content and upregulated the high affinity Pi transporters PHT1;2 and phosphorus starvation inducing (PSI) genes PHO2 and PHR1 under low Pi conditions. Transgenic Arabidopsis displayed tolerance to drought and salt stress by maintaining higher chlorophyll content and chlorophyll fluorescence, lower water loss rates, and ion leakage, which contributed to the survival of overexpression lines compared to the WT. Transcriptome profiling identified a peroxidase gene, POX, whose transcript was upregulated by these abiotic stresses. Furthermore, we confirmed that ZmARF1 bound to the auxin response element (AuxRE) in the promoter of POX and enhanced its transcription to mediate tolerance to oxidative stress imposed by low Pi, drought and salt stress in the transgenic seedlings. These results demonstrate that ZmARF1 has significant potential for improving the tolerance of crops to multiple abiotic stresses.

Dual functions of AreA, a GATA transcription factor, on influencing ganoderic acid biosynthesis in Ganoderma lucidum.

Nitrogen metabolism repression (NMR) has been well studied in filamentous fungi, but the molecular mechanism of its effects on fungal secondary metabolism has been generally unexplored. Ganoderic acid (GA) biosynthesis in Ganoderma lucidum differs between ammonia and nitrate nitrogen sources. To explain the functions of NMR in secondary metabolism, AreA, which is a core transcription factor of NMR, was characterized in G. lucidum. The transcription level of AreA was dramatically increased (approximately 4.5-folds), with the nitrate as the sole nitrogen source, compared with that with ammonia as the source. In addition, the expression of related genes involved in NMR was changed (upregulated of MeaB and downregulated of Nmr and GlnA) when AreA was knockdown. Yeast one-hybrid and electrophoretic mobility shift assay results showed that AreA could directly bind to the promoter of fps (encoding farnesyl-diphosphate synthase) to activate its expression. However, GA biosynthesis was increased (27% in the ammonia source and 77% in the nitrate source) in AreAi mutant strains versus that in control strains. These results showed that another important factor must participate in regulating GA biosynthesis other than the direct activation of AreA. Furthermore, we found that the content of nitric oxide (NO) was increased approximately 2.7-folds in the nitrate source compared with that in the ammonia. By adding the NO donor (SNP) or scavenger (cPTIO) and using NR-silenced or NR-overexpressed strains, we found that there was a negative correlation between the NO contents and GA biosynthesis. NO generated by nitrate reductase (NR) during the nitrogen utilization burst and could negatively influence GA biosynthesis. As a global transcription factor, AreA could also regulate the expression of NR. Our studies provide novel insight into the dual functions of AreA in GA biosynthesis during nitrogen assimilation.

14-3-3 proteins are involved in growth, hyphal branching, ganoderic acid biosynthesis, and response to abiotic stress in Ganoderma lucidum.

Ganoderma lucidum, which contains many pharmacologically active compounds, is regarded as a traditional medicinal fungus. Nevertheless, the scarcity of basic research limits the commercial value and utilization of G. lucidum. As a class of highly conserved, phosphopeptide-binding proteins present in all eukaryotes, 14-3-3 proteins play vital roles in controlling multiple physiological processes, including signal transduction, primary metabolism, and stress responses. However, knowledge of the roles of 14-3-3 proteins in Basidiomycetes is sparse. In this article, two homologs of 14-3-3 proteins, encoded by the two distinct genes GlBmh1 and GlBmh2, were distinguished in G. lucidum. We found that GlBmh1 and GlBmh2 were expressed at various developmental stages, including in vegetative mycelium cultivated on solid medium and in primordia and fruiting bodies. Moreover, we constructed GlBmh1 single-silenced strains, GlBmh2 single-silenced strains, and 14-3-3 double-silenced mutants for further study. When GlBmh1 and GlBmh2 were inhibited by RNA interference, the growth rate of mycelia was decreased, and the distance between the aerial hyphal branches was reduced; responses to various abiotic stresses such as oxidants and cell wall and osmotic stressors were also changed. Furthermore, the contents of secondary metabolite ganoderic acids (GAs) were increased after GlBmh1 and GlBmh2 were simultaneously silenced. Taken together, we provide evidence that implicates potential roles for the two 14-3-3 proteins in affecting growth and GA biosynthesis, thereby providing new insights into the basic functions of 14-3-3 proteins in G. lucidum.

Alternative oxidase impacts ganoderic acid biosynthesis by regulating intracellular ROS levels in Ganoderma lucidum.

The alternative oxidase (AOX), which forms a branch of the mitochondrial respiratory electron transport pathway, functions to sustain electron flux and alleviate reactive oxygen species (ROS) production. In this article, a homologous AOX gene was identified in Ganoderma lucidum. The coding sequence of the AOX gene in G. lucidum contains 1038 nucleotides and encodes a protein of 39.48 kDa. RNA interference (RNAi) was used to study the function of AOX in G. lucidum, and two silenced strains (AOXi6 and AOXi21) were obtained, showing significant decreases of approximately 60 and 50 %, respectively, in alternative pathway respiratory efficiency compared to WT. The content of ganoderic acid (GA) in the mutant strains AOXi6 and AOXi21 showed significant increases of approximately 42 and 44 %, respectively, compared to WT. Elevated contents of intermediate metabolites in GA biosynthesis and elevated transcription levels of corresponding genes were also observed in the mutant strains AOXi6 and AOXi21. In addition, the intracellular ROS content in strains AOXi6 and AOXi21 was significantly increased, by approximately 1.75- and 1.93-fold, respectively, compared with WT. Furthermore, adding N-acetyl-l-cysteine (NAC), a ROS scavenger, significantly depressed the intracellular ROS content and GA accumulation in AOX-silenced strains. These results indicate that AOX affects GA biosynthesis by regulating intracellular ROS levels. Our research revealed the important role of AOX in the secondary metabolism of G. lucidum.

The mitogen-activated protein kinase GlSlt2 regulates fungal growth, fruiting body development, cell wall integrity, oxidative stress and ganoderic acid biosynthesis in Ganoderma lucidum.

The mitogen-activated protein kinases (MAPKs) are crucial signaling instruments in eukaryotes that play key roles in regulating fungal growth, development, and secondary metabolism and in adapting to the environment. In this study, we characterized an Slt2-type MAPK in Ganoderma lucidum, GlSlt2, which was transcriptionally induced during the primordium and fruiting body stages. RNA interference was used to examine the function of GlSlt2. Knockdown of GlSlt2 caused defects in growth and increased hyphal branching as well as hypersensitivity to cell wall-disturbing substances. Consistently, the chitin and ß-1,3-d-glucan contents and the expression of cell wall biosynthesis genes were decreased and down-regulated, respectively, in GlSlt2 knockdown strains compared with those in the wild type (WT). In addition, no primordium or fruiting body could be observed in GlSlt2 knockdown strains. Furthermore, the intracellular reactive oxygen species (ROS) content and ganoderic acid biosynthesis also decreased in GlSlt2 knockdown strains. Addition of H2O2 could recover the decreased ganoderic acid content in GlSlt2 knockdown strains, indicating that GlSlt2 might regulate ganoderic acid biosynthesis via the intracellular ROS level. Overall, GlSlt2 is involved in hyphal growth, fruiting body development, cell wall integrity, oxidative stress and ganoderic acid biosynthesis in G. lucidum.

Semianalytical Modeling for Multiphase Flow in a Fractured Low-Permeability Gas Condensate Reservoir.

During the production of fractured low-permeability gas condensate reservoir (FLPGCR), a phase transition takes place in both the formation and wellbore, resulting in multiphase flow when the pressure drops below the dew point pressure. Additionally, the presence of fractures causes the formation of stress-sensitive characteristics. Nevertheless, traditional analytical models, such as the two-region model or three-region model, overlook the coupling impact of the above factors, which could lead to incorrect pressure transient response and erroneous estimation of well and formation parameters. Therefore, this work presents a semianalytical model for an FLPGCR considering the effects of multiphase flow, stress-sensitive, and wellbore phase redistribution. The gas condensate reservoir is divided into N banks, and the radial fluid saturation variation is modeled by multiple annular reservoirs with a constant saturation in each annular reservoir. The behavior of a fractured reservoir is modeled by using the dual-porosity model. The Pedrosa transform was utilized to address the nonlinear differential equation arising from stress-sensitive behavior. To verify the semianalytical solution, it was compared with numerical simulation results from CMG. The results showed that there are 10 flow regimes for the proposed model. The shape of the type curve has the potential to identify the degree of blockage within the FLPGCR. The wellbore phase redistribution only affects the first transitional-flow regime, which slows the rate of pressure drop. The stress sensitivity will lead to the upward characteristic of the curve in a later stage. More attention should be paid to the upward pressure derivative curve at late times, which is conventionally regarded as the effect of a closed boundary when it may not be the case. In addition, the shape factor and composite radius may obscure the radial flow regime. Finally, the proposed model was applied to interpret the pressure measurements recorded from the Bohai field in China, which exhibits a better fitting quality than the traditional models.

Pressure Transient Analysis for Hydraulically Fractured Wells with Changing Conductivity in Stratified Reservoirs: Case Study in Xinjiang Oilfield.

The oil development has been oriented toward deep-layer reservoirs and the commingling production and the separate-layer fracturing are important development methods. Currently, limited attention is given to the pressure transient analysis (PTA) of the fractured wells located in a stratified reservoir. Moreover, the proppant is very difficult to move inside the hydraulic fracture in the deep-layer reservoir, leading to the uneven fracture conductivity along the hydraulic fracture and increasing the complexity of PTA. To fill this gap, this work presented a fully analytical well test model for hydraulically fractured wells with changing fracture conductivity in stratified reservoirs, which is convenient to be used for interpreting the recorded pressure data from the oilfield due to its analytical nature. The establishment of this model is based on the trilinear flow model, Duhamel theorem, and pressure superposition principle. A systematic verification is conducted to ensure the validity of the proposed model. Furthermore, we offer a sensitivity analysis to investigate the effect of crucial parameters on pressure and pressure derivative, including the fracture extension, fracture conductivity, transmissibility factor, and storativity factor. Finally, a field case of a four-layer fractured well from Xinjiang Oilfield in Junggar Basin is interpreted to demonstrate the practicability of the presented model.

Functional analysis of an APSES transcription factor (GlSwi6) involved in fungal growth, fruiting body development and ganoderic-acid biosynthesis in Ganoderma lucidum.

The APSES transcription factors have been identified as key regulators of fungal development and other biological processes in fungi. In the present study, the function of Ganoderma lucidum GlSwi6, a homolog of Saccharomyces cerevisiae Swi6, was characterized. RNAi was used to examine the function of GlSwi6 in G. lucidum. Silencing GlSwi6 resulted in multiple developmental defects, including reduced fungal growth and increased hyphal branching, and the GlSwi6-silenced strains did not exhibit primordium or fruiting body formation. In addition, the H2O2 and ganoderic-acid (GA) levels of the GlSwi6-silenced strains decreased approximately 50% and 25%, respectively, compared with those of the WT strain. Furthermore, the addition of H2O2 led to the recovery of the GA levels of GlSwi6-silenced strains, implying that GlSwi6 might regulate GA biosynthesis by regulating the intracellular ROS levels. Taken together, these results indicate that GlSwi6 is involved in fungal growth, development and GA biosynthesis in G. lucidum.

Heat stress-induced reactive oxygen species participate in the regulation of HSP expression, hyphal branching and ganoderic acid biosynthesis in Ganoderma lucidum.

Heat stress (HS) is an important environmental factor that affects the growth and metabolism of edible fungi, but the molecular mechanism of the heat stress response (HSR) remains unclear. We previously reported that HS treatment increased the length between two hyphal branches and induced the accumulation of ganoderic acid biosynthesis and the gene expression of heat shock proteins (HSPs) in Ganoderma lucidum. In this study, we found that HS induced a significant increase in the cytosolic ROS concentration, and exogenously added ROS scavengers NAC, VC and NADPH oxidase (Nox) inhibitor DPI reduce the cytosolic ROS accumulation in G. lucidum. In addition, the phenomena of the increased gene expression and increased length between the two hyphal branches and the accumulation of GA biosynthesis induced by HS were mitigated. Furthermore, we investigated the effects of HS on Nox-silenced strains (NoxABi-10, NoxABi-11 and NoxRi-4, NoxRi-7) and found that the level of ROS concentration was lower than that in wild-type (WT) strains treated with HS. Additionally, Nox silenced strains reduced the HS-induced increase in HSP expression, the length between two hyphal branches and GA biosynthesis compared with the WT strain. These data indicate that HS-induced ROS participate in the regulation of HSP expression, hyphal branching and ganoderic acid biosynthesis in G. lucidum. In addition, these findings identified potential pathways linking ROS networks to HSR, physiological and metabolic processes in fungi and provide a valuable reference for studying the role of ROS in HSR, mycelium growth and secondary metabolites.