Metabolic remodeling by RNA polymerase gene mutations is associated with reduced ß-lactam susceptibility in oxacillin-susceptible MRSA.

The emergence of oxacillin-susceptible methicillin-resistant Staphylococcus aureus (OS-MRSA) has imposed further challenges to the clinical management of MRSA infections. When exposed to ß-lactam antibiotics, these strains can easily acquire reduced ß-lactam susceptibility through chromosomal mutations, including those in RNA polymerase (RNAP) genes such as rpoBC, which may then lead to treatment failure. Despite the increasing prevalence of such strains and the apparent challenges they pose for diagnosis and treatment, there is limited information available on the actual mechanisms underlying such chromosomal mutation-related transitions to reduced ß-lactam susceptibility, as it does not directly associate with the expression of mecA. This study investigated the cellular physiology and metabolism of six missense mutants with reduced oxacillin susceptibility, each carrying respective mutations on RpoBH929P, RpoBQ645H, RpoCG950R, RpoCG498D, RpiAA64E, and FruBA211E, using capillary electrophoresis-mass spectrometry-based metabolomics analysis. Our results showed that rpoBC mutations caused RNAP transcription dysfunction, leading to an intracellular accumulation of ribonucleotides. These mutations also led to the accumulation of UDP-Glc/Gal and UDP-GlcNAc, which are precursors of UTP-associated peptidoglycan and wall teichoic acid. Excessive amounts of building blocks then contributed to the cell wall thickening of mutant strains, as observed in transmission electron microscopy, and ultimately resulted in decreased susceptibility to ß-lactam in OS-MRSA. IMPORTANCE: The emergence of oxacillin-susceptible methicillin-resistant Staphylococcus aureus (OS-MRSA) strains has created new challenges for treating MRSA infections. These strains can become resistant to ß-lactam antibiotics through chromosomal mutations, including those in the RNA polymerase (RNAP) genes such as rpoBC, leading to treatment failure. This study investigated the mechanisms underlying reduced ß-lactam susceptibility in four rpoBC mutants of OS-MRSA. The results showed that rpoBC mutations caused RNAP transcription dysfunction, leading to an intracellular accumulation of ribonucleotides and precursors of peptidoglycan as well as wall teichoic acid. This, in turn, caused thickening of the cell wall and ultimately resulted in decreased susceptibility to ß-lactam in OS-MRSA. These findings provide insights into the mechanisms of antibiotic resistance in OS-MRSA and highlight the importance of continued research in developing effective treatments to combat antibiotic resistance.

Natural variation of TBR confers plant zinc toxicity tolerance through root cell wall pectin methylesterification.

Zinc (Zn) is an essential micronutrient but can be cytotoxic when present in excess. Plants have evolved mechanisms to tolerate Zn toxicity. To identify genetic loci responsible for natural variation of plant tolerance to Zn toxicity, we conduct genome-wide association studies for root growth responses to high Zn and identify 21 significant associated loci. Among these loci, we identify Trichome Birefringence (TBR) allelic variation determining root growth variation in high Zn conditions. Natural alleles of TBR determine TBR transcript and protein levels which affect pectin methylesterification in root cell walls. Together with previously published data showing that pectin methylesterification increase goes along with decreased Zn binding to cell walls in TBR mutants, our findings lead to a model in which TBR allelic variation enables Zn tolerance through modulating root cell wall pectin methylesterification. The role of TBR in Zn tolerance is conserved across dicot and monocot plant species.

Unraveling the antifungal and anti-aflatoxin B1 mechanisms of piperitone on Aspergillus flavus.

Aspergillus flavus infects important crops and produces carcinogenic aflatoxins, posing a serious threat to food safety and human health. Biochemical analysis and RNA-seq were performed to investigate the effects and mechanisms of piperitone on A. flavus growth and aflatoxin B1 biosynthesis. Piperitone significantly inhibited the growth of A. flavus, AFB1 production, and its pathogenicity on peanuts and corn flour. Differentially expressed genes (DEGs) associated with the synthesis of chitin, glucan, and ergosterol were markedly down-regulated, and the ergosterol content was reduced, resulting in a disruption in the integrity of the cell wall and cell membrane. Moreover, antioxidant genes were down-regulated, the correspondingly activities of antioxidant enzymes such as catalase, peroxidase, and superoxide dismutase were reduced, and levels of superoxide anion and hydrogen peroxide were increased, leading to a burst of reactive oxygen species (ROS). Accompanied by ROS accumulation, DNA fragmentation and cell autophagy were observed, and 16 aflatoxin cluster genes were down-regulated. Overall, piperitone disrupts the integrity of the cell wall and cell membrane, triggers the accumulation of ROS, causes DNA fragmentation and cell autophagy, ultimately leading to defective growth and impaired AFB1 biosynthesis.

Toxic eburicol accumulation drives the antifungal activity of azoles against Aspergillus fumigatus.

Azole antifungals inhibit the sterol C14-demethylase (CYP51/Erg11) of the ergosterol biosynthesis pathway. Here we show that the azole-induced synthesis of fungicidal cell wall carbohydrate patches in the pathogenic mold Aspergillus fumigatus strictly correlates with the accumulation of the CYP51 substrate eburicol. A lack of other essential ergosterol biosynthesis enzymes, such as sterol C24-methyltransferase (Erg6A), squalene synthase (Erg9) or squalene epoxidase (Erg1) does not trigger comparable cell wall alterations. Partial repression of Erg6A, which converts lanosterol into eburicol, increases azole resistance. The sterol C5-desaturase (ERG3)-dependent conversion of eburicol into 14-methylergosta-8,24(28)-dien-3ß,6α-diol, the "toxic diol" responsible for the fungistatic activity against yeasts, is not required for the fungicidal effects in A. fumigatus. While ERG3-lacking yeasts are azole resistant, ERG3-lacking A. fumigatus becomes more susceptible. Mutants lacking mitochondrial complex III functionality, which are much less effectively killed, but strongly inhibited in growth by azoles, convert eburicol more efficiently into the supposedly "toxic diol". We propose that the mode of action of azoles against A. fumigatus relies on accumulation of eburicol which exerts fungicidal effects by triggering cell wall carbohydrate patch formation.

Cell Wall Binding Strategies Based on Cu3SbS3 Nanoparticles for Selective Bacterial Elimination and Promotion of Infected Wound Healing.

Utilizing nanomaterials as an alternative to antibiotics, with a focus on maintaining high biosafety, has emerged as a promising strategy to combat antibiotic resistance. Nevertheless, the challenge lies in the indiscriminate attack of nanomaterials on both bacterial and mammalian cells, which limits their practicality. Herein, Cu3SbS3 nanoparticles (NPs) capable of generating reactive oxygen species (ROS) are discovered to selectively adsorb and eliminate bacteria without causing obvious harm to mammalian cells, thanks to the interaction between O of N-acetylmuramic acid in bacterial cell walls and Cu of the NPs. Coupled with the short diffusion distance of ROS in the surrounding medium, a selective antibacterial effect is achieved. Additionally, the antibacterial mechanism is then identified: Cu3SbS3 NPs catalyze the generation of O2•-, which has subsequently been conversed by superoxide dismutase to H2O2. The latter is secondary catalyzed by the NPs to form •OH and 1O2, initiating an in situ attack on bacteria. This process depletes bacterial glutathione in conjunction with the disruption of the antioxidant defense system of bacteria. Notably, Cu3SbS3 NPs are demonstrated to efficiently impede biofilm formation; thus, a healing of MRSA-infected wounds was promoted. The bacterial cell wall-binding nanoantibacterial agents can be widely expanded through diversified design.

Melatonin Increases Root Cell Wall Phosphorus Reutilization via an NO Dependent Pathway in Rice (Oryza sativa).

Melatonin (MT) has been implicated in the plant response to phosphorus (P) stress; however, the precise molecular mechanisms involved remain unclear. This study investigated whether MT controls internal P distribution and root cell wall P remobilization in rice. Rice was treated with varying MT and P levels and analyzed using biochemical and molecular techniques to study phosphorus utilization. The results demonstrated that low P levels lead to a rapid increase in endogenous MT levels in rice roots. Furthermore, the exogenous application of MT significantly improved rice tolerance to P deficiency, as evidenced by the increased biomass and reduced proportion of roots to shoots under P-deficient conditions. MT application also mitigated the decrease in P content regardless in both the roots and shoots. Mechanistically, MT accelerated the reutilization of P, particularly in the root pectin fraction, leading to increased soluble P liberation. In addition, MT enhanced the expression of OsPT8, a gene involved in root-to-shoot P translocation. Furthermore, we observed that MT induced the production of nitric oxide (NO) in P-deficient rice roots and that the mitigating effect of MT on P deficiency was compromised in the presence of the NO inhibitor, c-PTIO, implying that NO is involved in the MT-facilitated mitigation of P deficiency in rice. Overall, our findings highlight the potential of MT as a promising strategy for enhancing rice tolerance to P deficiency and improving P use efficiency in agricultural practices.

Adaptative survival of Aspergillus fumigatus to echinocandins arises from cell wall remodeling beyond ß-1,3-glucan synthesis inhibition.

Antifungal echinocandins inhibit the biosynthesis of ß-1,3-glucan, a major and essential polysaccharide component of the fungal cell wall. However, the efficacy of echinocandins against the pathogen Aspergillus fumigatus is limited. Here, we use solid-state nuclear magnetic resonance (ssNMR) and other techniques to show that echinocandins induce dynamic changes in the assembly of mobile and rigid polymers within the A. fumigatus cell wall. The reduction of ß-1,3-glucan induced by echinocandins is accompanied by a concurrent increase in levels of chitin, chitosan, and highly polymorphic α-1,3-glucans, whose physical association with chitin maintains cell wall integrity and modulates water permeability. The rearrangement of the macromolecular network is dynamic and controls the permeability and circulation of the drug throughout the cell wall. Thus, our results indicate that echinocandin treatment triggers compensatory rearrangements in the cell wall that may help A. fumigatus to tolerate the drugs' antifungal effects.

The trichothecene mycotoxin deoxynivalenol facilitates cell-to-cell invasion during wheat-tissue colonization by Fusarium graminearum.

Fusarium head blight disease on small-grain cereals is primarily caused by the ascomycete fungal pathogen Fusarium graminearum. Infection of floral spike tissues is characterized by the biosynthesis and secretion of potent trichothecene mycotoxins, of which deoxynivalenol (DON) is widely reported due to its negative impacts on grain quality and consumer safety. The TRI5 gene encodes an essential enzyme in the DON biosynthesis pathway and the single gene deletion mutant, ΔTri5, is widely reported to restrict disease progression to the inoculated spikelet. In this study, we present novel bioimaging evidence revealing that DON facilitates the traversal of the cell wall through plasmodesmata, a process essential for successful colonization of host tissue. Chemical complementation of ΔTri5 did not restore macro- or microscopic phenotypes, indicating that DON secretion is tightly regulated both spatially and temporally. A comparative qualitative and quantitative morphological cellular analysis revealed infections had no impact on plant cell wall thickness. Immunolabelling of callose at plasmodesmata during infection indicates that DON can increase deposits when applied exogenously but is reduced when F. graminearum hyphae are present. This study highlights the complexity of the interconnected roles of mycotoxin production, cell wall architecture and plasmodesmata in this highly specialized interaction.

Activation of the cell wall integrity pathway negatively regulates TORC2-Ypk1/2 signaling through blocking eisosome disassembly in Saccharomyces cerevisiae.

The target of rapamycin complex 2 (TORC2) signaling is associated with plasma membrane (PM) integrity. In Saccharomyces cerevisiae, TORC2-Ypk1/2 signaling controls sphingolipid biosynthesis, and Ypk1/2 phosphorylation by TORC2 under PM stress conditions is increased in a Slm1/2-dependent manner, under which Slm1 is known to be released from an eisosome, a furrow-like invagination PM structure. However, it remains unsolved how the activation machinery of TORC2-Ypk1/2 signaling is regulated. Here we show that edelfosine, a synthetic lysophospholipid analog, inhibits the activation of TORC2-Ypk1/2 signaling, and the cell wall integrity (CWI) pathway is involved in this inhibitory effect. The activation of CWI pathway blocked the eisosome disassembly promoted by PM stress and the release of Slm1 from eisosomes. Constitutive activation of TORC2-Ypk1/2 signaling exhibited increased sensitivity to cell wall stress. We propose that the CWI pathway negatively regulates the TORC2-Ypk1/2 signaling, which is involved in the regulatory mechanism to ensure the proper stress response to cell wall damage.

Halenaquinol Blocks Staphylococcal Protein A Anchoring on Cell Wall Surface by Inhibiting Sortase A in Staphylococcus aureus.

Sortase A (SrtA) is a cysteine transpeptidase that binds to the periplasmic membrane and plays a crucial role in attaching surface proteins, including staphylococcal protein A (SpA), to the peptidoglycan cell wall. Six pentacyclic polyketides (1-6) were isolated from the marine sponge Xestospongia sp., and their structures were elucidated using spectroscopic techniques and by comparing them to previously reported data. Among them, halenaquinol (2) was found to be the most potent SrtA inhibitor, with an IC50 of 13.94 µM (4.66 µg/mL). Semi-quantitative reverse transcription PCR data suggest that halenaquinol does not inhibit the transcription of srtA and spA, while Western blot analysis and immunofluorescence microscopy images suggest that it blocks the cell wall surface anchoring of SpA by inhibiting the activity of SrtA. The onset and magnitude of the inhibition of SpA anchoring on the cell wall surface in S. aureus that has been treated with halenaquinol at a value 8× that of the IC50 of SrtA are comparable to those for an srtA-deletion mutant. These findings contribute to the understanding of the mechanism by which marine-derived pentacyclic polyketides inhibit SrtA, highlighting their potential as anti-infective agents targeting S. aureus virulence.

Structure and in vitro antiproliferative activity against breast cancer cells of the cell-wall polysaccharide from the marine bacterium Kangiella japonica KMM 3899T.

Kangiella japonica KMM 3899T is a Gram-negative bacterium isolated from a sandy sediment sample collected from the Sea of Japan. Here the results of the structure and the biological activity against breast cancer cells of the cell-wall polysaccharide from K. japonica KMM 3899T have been described. The structure of the repeating unit of the polysaccharide was elucidated using chemical analysis and NMR spectroscopy: →4)-α-L-GalpNAc3AcA-(1 â†’ 3)-α-D-GlcpNAc-(1 â†’ 4)-ß-D-GlcpNAc3NAcAN-(1→. The cell-wall polysaccharide had an antiproliferative effect against T-47D cells. Flow cytometric and Western blot analysis revealed that the polysaccharide induced S phase arrest and mitochondrial-dependent apoptosis.

Guarding the walls: the multifaceted roles of Bce modules in cell envelope stress sensing and antimicrobial resistance.

Bacteria have developed diverse strategies for defending their cell envelopes from external threats. In Firmicutes, one widespread strategy is to use Bce modules-membrane protein complexes that unite a peptide-detoxifying ABC transporter with a stress response coordinating two-component system. These modules provide specific, front-line defense for a wide variety of antimicrobial peptides and small molecule antibiotics as well as coordinate responses for heat, acid, and oxidative stress. Because of these abilities, Bce modules play important roles in virulence and the development of antibiotic resistance in a variety of pathogens, including Staphylococcus, Streptococcus, and Enterococcus species. Despite their importance, Bce modules are still poorly understood, with scattered functional data in only a small number of species. In this review, we will discuss Bce module structure in light of recent cryo-electron microscopy structures of the B. subtilis BceABRS module and explore the common threads and variations-on-a-theme in Bce module mechanisms across species. We also highlight the many remaining questions about Bce module function. Understanding these multifunctional membrane complexes will enhance our understanding of bacterial stress sensing and may point toward new therapeutic targets for highly resistant pathogens.

A potential space-making role in cell wall biogenesis for SltB1and DacB revealed by a beta-lactamase induction phenotype in Pseudomonas aeruginosa.

Pseudomonas aeruginosa encodes the beta-lactamase AmpC, which promotes resistance to beta-lactam antibiotics. Expression of ampC is induced by anhydro-muropeptides (AMPs) released from the peptidoglycan (PG) cell wall upon beta-lactam treatment. AmpC can also be induced via genetic inactivation of PG biogenesis factors such as the endopeptidase DacB that cleaves PG crosslinks. Mutants in dacB occur in beta-lactam-resistant clinical isolates of P. aeruginosa, but it has remained unclear why DacB inactivation promotes ampC induction. Similarly, the inactivation of lytic transglycosylase (LT) enzymes such as SltB1 that cut PG glycans has also been associated with ampC induction and beta-lactam resistance. Given that LT enzymes are capable of producing AMP products that serve as ampC inducers, this latter observation has been especially difficult to explain. Here, we show that ampC induction in sltB1 or dacB mutants requires another LT enzyme called MltG. In Escherichia coli, MltG has been implicated in the degradation of nascent PG strands produced upon beta-lactam treatment. Accordingly, in P. aeruginosa sltB1 and dacB mutants, we detected the MltG-dependent production of pentapeptide-containing AMP products that are signatures of nascent PG degradation. Our results therefore support a model in which SltB1 and DacB use their PG-cleaving activity to open space in the PG matrix for the insertion of new material. Thus, their inactivation mimics low-level beta-lactam treatment by reducing the efficiency of new PG insertion into the wall, causing the degradation of some nascent PG material by MltG to produce the ampC-inducing signal. IMPORTANCE: Inducible beta-lactamases like the ampC system of Pseudomonas aeruginosa are a common determinant of beta-lactam resistance among gram-negative bacteria. The regulation of ampC is elegantly tuned to detect defects in cell wall synthesis caused by beta-lactam drugs. Studies of mutations causing ampC induction in the absence of drug therefore promise to reveal new insights into the process of cell wall biogenesis in addition to aiding our understanding of how resistance to beta-lactam antibiotics arises in the clinic. In this study, the ampC induction phenotype for mutants lacking a glycan-cleaving enzyme or an enzyme that cuts cell wall crosslinks was used to uncover a potential role for these enzymes in making space in the wall matrix for the insertion of new material during cell growth.

Potent and specific antibiotic combination therapy against Clostridioides difficile.

Keratinicyclins and keratinimicins are recently discovered glycopeptide antibiotics. Keratinimicins show broad-spectrum activity against Gram-positive bacteria, while keratinicyclins form a new chemotype by virtue of an unusual oxazolidinone moiety and exhibit specific antibiosis against Clostridioides difficile. Here we report the mechanism of action of keratinicyclin B (KCB). We find that steric constraints preclude KCB from binding peptidoglycan termini. Instead, KCB inhibits C. difficile growth by binding wall teichoic acids (WTAs) and interfering with cell wall remodeling. A computational model, guided by biochemical studies, provides an image of the interaction of KCB with C. difficile WTAs and shows that the same H-bonding framework used by glycopeptide antibiotics to bind peptidoglycan termini is used by KCB for interacting with WTAs. Analysis of KCB in combination with vancomycin (VAN) shows highly synergistic and specific antimicrobial activity, and that nanomolar combinations of the two drugs are sufficient for complete growth inhibition of C. difficile, while leaving common commensal strains unaffected.

Calcium polypeptide mitigates Cd toxicity in rice via reducing oxidative stress and regulating pectin modification.

KEY MESSAGE: Calcium polypeptide plays a key role during cadmium stress responses in rice, which is involved in increasing peroxidase activity, modulating pectin methylesterase activity, and regulating cell wall by reducing malondialdehyde content. Cadmium (Cd) contamination threatens agriculture and human health globally, emphasizing the need for sustainable methods to reduce cadmium toxicity in crops. Calcium polypeptide (CaP) is a highly water-soluble small molecular peptide acknowledged for its potential as an organic fertilizer in promoting plant growth. However, it is still unknown whether CaP has effects on mitigating Cd toxicity. Here, we investigated the effect of CaP application on the ability to tolerate toxic Cd in rice. We evaluated the impact of CaP on rice seedlings under varying Cd stress conditions and investigated the effect mechanism of CaP mitigating Cd toxicity by Fourier transform infrared spectroscopy (FTIR), fluorescent probe dye, immunofluorescent labeling, and biochemical analysis. We found a notable alleviation of Cd toxicity by reduced malondialdehyde content and increased peroxidase activity. In addition, our findings reveal that CaP induces structural alterations in the root cell wall by modulating pectin methylesterase activity. Altogether, our results confirm that CaP not only promoted biomass accumulation but also reduced Cd concentration in rice. This study contributes valuable insights to sustainable strategies for addressing Cd contamination in agricultural ecosystems.

Mechanism of the antimicrobial activity induced by phosphatase inhibitor sodium ortho-vanadate.

The present study describes a novel antimicrobial mechanism based on Sodium Orthovanadate (SOV), an alkaline phosphatase inhibitor. Scanning electron microscopy (SEM), transmission electron microscopy (TEM) and atomic force microscopy (AFM) were employed to examine the surface morphologies of the test organism, Escherichia coli (E. coli), during various antibacterial phases. Our results indicated that SOV kills bacteria by attacking cell wall growth and development, leaving E. coli's outer membrane intact. Our antimicrobial test indicated that the MIC of SOV for both E. coli and Lactococcus lactis (L. lactis) is 40 µM. A combination of quantum mechanical calculations and vibrational spectroscopy revealed that divanadate from SOV strongly coordinates with Ca2+ and Mg2+, which are the activity centers for the phosphatase that regulates bacterial cell wall synthesis. The current study is the first to propose the antibacterial mechanism caused by SOV attacking cell wall.

Controlled delivery of nikkomycin by PEG coated PLGA nanoparticles inhibits chitin synthase to prevent growth of Aspergillus flavus and Aspergillus fumigatus.

Aspergillosis is one of the most common fungal infections that can threaten individuals with immune compromised condition. Due to the increasing resistance of pathogens to the existing antifungal drugs, it is difficult to tackle such disease conditions. Whereas, nikkomycin is an emerging safe and effective antifungal drug which causes fungal cell wall disruption by inhibiting chitin synthase. Hence, the study aims at the development of nikkomycin loaded PEG coated PLGA nanoparticles for its increased antifungal efficiency and inhibiting Aspergillus infections. The P-PLGA-Nik NPs were synthesized by w/o/w double emulsification method which resulted in a particle size of 208.3 ± 15 nm with a drug loading of 52.97 %. The NPs showed first order diffusion-controlled drug release which was sustained for 24 h. These nanoparticle's antifungal efficacy was tested using the CLSI - M61 guidelines and the MIC50 defined against Aspergillus flavus and Aspergillus fumigatus was found to be >32 µg/ml which was similar to the nikkomycin MIC. The hyphal tip bursting showed the fungal cell wall disruption. The non-cytotoxic and non-haemolytic nature highlights the drug safety profile.

Methyl jasmonate enhances the safe production ability of Cd-stressed wheat by regulating the antioxidant capacity, Cd absorption, and distribution in wheat.

Identifying green and effective measures for reducing wheat Cd toxicity and grain Cd accumulation is crucial. This study used seedling sand culture and full-grown pot experiments of wheat cultivars 'Luomai23' (LM) and 'Zhongyu10' (ZY). The purpose was to determine the effects of exogenous MeJA on the phenotype, photosynthesis, antioxidant system, Cd accumulation and distribution, transporter gene expression, and cell wall properties of Cd-stressed wheat. Compared with Cd treatment alone, the plant height and maximum root length treated with 0.001 µM MeJA increased by more than 6.3% and 16.6%, respectively. Under 5 mg⋅kg-1 Cd treatment, spraying 10 µM MeJA increased the photosynthetic rate of LM and ZY by 23.5% and 35.8% at the filling stage, respectively. Methyl jasmonate significantly reduced the H2O2 and MDA contents by increasing the activities of POD, DHAR, MDHAR, and GR and the contents of AsA and GSH. Applicating MeJA increased the content of chelate substances, cell wall polysaccharides, and cell wall functional groups. Besides, MeJA regulated the expression of Cd transporter genes, with shoot and root Cd content decreasing by 46.7% and 27.9% in LM, respectively. Spraying 10 µM MeJA reduced Cd absorption and translocation from vegetative organs to grains, thus reducing the grain Cd content of LM and ZY by 36.1 and 39.9% under 5 mg⋅kg-1 Cd treatment, respectively. Overexpressing TaJMT significantly increased the MeJA content and Cd tolerance of Arabidopsis. These results have improved the understanding of the mechanism through which MeJA alleviates Cd toxicity and reduces Cd accumulation in wheat.

Impacts of in-vitro zebularine treatment on genome-wide DNA methylation and transcriptomic profiles in Salix purpurea L.

Renewable energy resources such as biomass are crucial for a sustainable global society. Trees are a major source of lignocellulosic biomass, which can vary in response to different environmental factors owing to epigenetic regulation, such as DNA C-methylation. To investigate the effects of DNA methylation on plant development and wood formation, and its impacts on gene expression, with a focus on secondary cell wall (SCW)-associated genes, Salix purpurea plantlets were cloned from buds derived from a single hybrid tree for both treatment and control conditions. For the treatment condition, buds were exposed to 50 µM zebularine in vitro and a combined strategy of whole-genome bisulfite sequencing (WGBS) and RNA-seq was employed to examine the methylome and transcriptome profiles of different tissues collected at various time points under both conditions. Transcriptomic and methylome data revealed that most of the promoter and gene body demethylation had no marked effects on the expression profiles of genes. Nevertheless, gene expression tended to decrease with the increased methylation levels of genes with highly methylated promoters. Results indicated that demethylation is less evident in centromeric regions and sex chromosomes. Promoters of secondary cell wall-associated genes, such as 4-coumarate-CoA ligase-like and Rac-like GTP-binding protein RHO, were differentially methylated in the secondary xylem samples collected from two-month potted treated plants compared to control samples. Our results provide novel insights into DNA methylation and gene expression landscapes and a basis for investigating the epigenetic regulation of wood formation in S. purpurea as a model plant for bioenergy species.

The different response of Brassica napus genotypes to microspore embryogenesis induced by heat shock and trichostatin A is not determined by changes in cell wall structure and composition but by different stress tolerance.

During microspore embryogenesis, microspores are induced to develop into haploid embryos. In Brassica napus, microspore embryogenesis is induced by a heat shock (HS), which initially produces embryogenic structures with different cell wall architectures and compositions, and with different potentials to develop into embryos. The B. napus DH4079 and DH12075 genotypes have high and very low embryo yields, respectively. In DH12075, embryo yield is greatly increased by combining HS and the histone deacetylase (HDAC) inhibitor trichostatin A (TSA). However, we show that HS + TSA inhibits embryogenesis in the highly embryogenic DH4079 line. To ascertain why TSA has such different effects in these lines, we treated DH4079 and DH12075 microspore cultures with TSA and compared the cell wall structure and composition of the different embryogenic structures in both lines, specifically the in situ levels and distribution of callose, cellulose, arabinogalactan proteins and high and low methyl-esterified pectin. For both lines, HS + TSA led to the formation of cell walls unfavorable for embryogenesis progression, with reduced levels of arabinogalactan proteins, reduced cell adhesion of inner walls and altered pectin composition. Thus, TSA effects on cell walls cannot explain their different embryogenic response to TSA. We also applied TSA to DH4079 cultures at different times and concentrations before HS application, with no negative effects on embryogenic induction. These results indicate that DH4079 microspores are hypersensitive to combined TSA and HS treatments, and open up new hypotheses about the causes of such hypersensitivity.