The Characterization of a Novel PrMADS11 Transcription Factor from Pinus radiata Induced Early in Bent Pine Stem.

A novel MADS-box transcription factor from Pinus radiata D. Don was characterized. PrMADS11 encodes a protein of 165 amino acids for a MADS-box transcription factor belonging to group II, related to the MIKC protein structure. PrMADS11 was differentially expressed in the stems of pine trees in response to 45° inclination at early times (1 h). Arabidopsis thaliana was stably transformed with a 35S::PrMADS11 construct in an effort to identify the putative targets of PrMADS11. A massive transcriptome analysis revealed 947 differentially expressed genes: 498 genes were up-regulated, and 449 genes were down-regulated due to the over-expression of PrMADS11. The gene ontology analysis highlighted a cell wall remodeling function among the differentially expressed genes, suggesting the active participation of cell wall modification required during the response to vertical stem loss. In addition, the phenylpropanoid pathway was also indicated as a PrMADS11 target, displaying a marked increment in the expression of the genes driven to the biosynthesis of monolignols. The EMSA assays confirmed that PrMADS11 interacts with CArG-box sequences. This TF modulates the gene expression of several molecular pathways, including other TFs, as well as the genes involved in cell wall remodeling. The increment in the lignin content and the genes involved in cell wall dynamics could be an indication of the key role of PrMADS11 in the response to trunk inclination.

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.

Expression profile analysis of cotton fiber secondary cell wall thickening stage.

To determine the genes associated with the fiber strength trait in cotton, three different cotton cultivars were selected: Sea Island cotton (Xinhai 32, with hyper-long fibers labeled as HL), and upland cotton (17-24, with long fibers labeled as L, and 62-33, with short fibers labeled as S). These cultivars were chosen to assess fiber samples with varying qualities. RNA-seq technology was used to analyze the expression profiles of cotton fibers at the secondary cell wall (SCW) thickening stage (20, 25, and 30 days post-anthesis (DPA)). The results showed that a large number of differentially expressed genes (DEGs) were obtained from the three assessed cotton cultivars at different stages of SCW development. For instance, at 20 DPA, Sea Island cotton (HL) had 6,215 and 5,364 DEGs compared to upland cotton 17-24 (L) and 62-33 (S), respectively. Meanwhile, there were 1,236 DEGs between two upland cotton cultivars, 17-24 (L) and 62-33 (S). Gene Ontology (GO) term enrichment identified 42 functions, including 20 biological processes, 11 cellular components, and 11 molecular functions. Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analysis identified several pathways involved in SCW synthesis and thickening, such as glycolysis/gluconeogenesis, galactose metabolism, propanoate metabolism, biosynthesis of unsaturated fatty acids pathway, valine, leucine and isoleucine degradation, fatty acid elongation pathways, and plant hormone signal transduction. Through the identification of shared DEGs, 46 DEGs were found to exhibit considerable expressional differences at different fiber stages from the three cotton cultivars. These shared DEGs have functions including REDOX enzymes, binding proteins, hydrolases (such as GDSL thioesterase), transferases, metalloproteins (cytochromatin-like genes), kinases, carbohydrates, and transcription factors (MYB and WRKY). Therefore, RT-qPCR was performed to verify the expression levels of nine of the 46 identified DEGs, an approach which demonstrated the reliability of RNA-seq data. Our results provided valuable molecular resources for clarifying the cell biology of SCW biosynthesis during fiber development in cotton.

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.

The impact of mechanical stress on anatomy, morphology, and gene expression in Urtica dioica L.

MAIN CONCLUSION: Mechanical stress induces distinct anatomical, molecular, and morphological changes in Urtica dioica, affecting trichome development, gene expression, and leaf morphology under controlled conditions The experiments were performed on common nettle, a widely known plant characterized by high variability of leaf morphology and responsiveness to mechanical touch. A specially constructed experimental device was used to study the impact of mechanical stress on Urtica dioica plants under strictly controlled parameters of the mechanical stimulus (touching) and environment in the growth chamber. The general anatomical structure of the plants that were touched was similar to that of control plants, but the shape of the internodes' cross section was different. Stress-treated plants showed a distinct four-ribbed structure. However, as the internodes progressed, the shape gradually approached a rectangular form. The epidermis of control plants included stinging, glandular and simple setulose trichomes, but plants that were touched had no stinging trichomes, and setulose trichomes accumulated more callose. Cell wall lignification occurred in the older internodes of the control plants compared to stress-treated ones. Gene analysis revealed upregulation of the expression of the UdTCH1 gene in touched plants compared to control plants. Conversely, the expression of UdERF4 and UdTCH4 was downregulated in stressed plants. These data indicate that the nettle's response to mechanical stress reaches the level of regulatory networks of gene expression. Image analysis revealed reduced leaf area, increased asymmetry and altered contours in touched leaves, especially in advanced growth stages, compared to control plants. Our results indicate that mechanical stress triggers various anatomical, molecular, and morphological changes in nettle; however, further interdisciplinary research is needed to better understand the underlying physiological mechanisms.

Exploring the plasmodesmata callose-binding protein gene family in upland cotton: unraveling insights for enhancing fiber length.

Plasmodesmata are transmembrane channels embedded within the cell wall that can facilitate the intercellular communication in plants. Plasmodesmata callose-binding (PDCB) protein that associates with the plasmodesmata contributes to cell wall extension. Given that the elongation of cotton fiber cells correlates with the dynamics of the cell wall, this protein can be related to the cotton fiber elongation. This study sought to identify PDCB family members within the Gossypium. hirsutum genome and to elucidate their expression profiles. A total of 45 distinct family members were observed through the identification and screening processes. The analysis of their physicochemical properties revealed the similarity in the amino acid composition and molecular weight across most members. The phylogenetic analysis facilitated the construction of an evolutionary tree, categorizing these members into five groups mainly distributed on 20 chromosomes. The fine mapping results facilitated a tissue-specific examination of group V, revealing that the expression level of GhPDCB9 peaked five days after flowering. The VIGS experiments resulted in a marked decrease in the gene expression level and a significant reduction in the mature fiber length, averaging a shortening of 1.43-4.77 mm. The results indicated that GhPDCB9 played a pivotal role in the cotton fiber development and served as a candidate for enhancing cotton yield.

The transcriptional regulation of a putative hemicellulose gene, PtrPARVUS2 in poplar.

The plant cell wall serves as a critical interface between the plant and its environment, offering protection against various stresses and contributing to biomass production. Hemicellulose is one of the major components of the cell wall, and understanding the transcriptional regulation of its production is essential to fully understanding cell wall formation. This study explores the regulatory mechanisms underlying one of the genes involved in hemicellulose biosynthesis, PtrPARVUS2. Six transcription factors (TFs) were identified from a xylem-biased library to negatively regulate PtrPARVUS2 expression. These TFs, belonging to diverse TF families, were confirmed to bind to specific cis-elements in the PtrPARVUS2 promoter region, as validated by Yeast One-Hybrid (Y1H) assays, transient expression analysis, and Chromatin Immunoprecipitation sequencing (ChIP-seq) assays. Furthermore, motif analysis identified putative cis-regulatory elements bound by these TFs, shedding light on the transcriptional regulation of SCW biosynthesis genes. Notably, several TFs targeted genes encoding uridine diphosphate glycosyltransferases (UGTs), crucial enzymes involved in hemicellulose glycosylation. Phylogenetic analysis of UGTs regulated by these TFs highlighted their diverse roles in modulating hemicellulose synthesis. Overall, this study identifies a set of TFs that regulate PARVUS2 in poplar, providing insights into the intricate coordination of TFs and PtrPARVUS2 in SCW formation. Understanding these regulatory mechanisms enhances our ability to engineer plant biomass for tailored applications, including biofuel production and bioproduct development.

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.

Roles of the Arabidopsis KEULE Gene in Postembryonic Development.

Cytokinesis in plant cells begins with the fusion of vesicles that transport cell wall materials to the center of the cell division plane, where the cell plate forms and expands radially until it fuses with the parental cell wall. Vesicle fusion is facilitated by trans-SNARE complexes, with assistance from Sec1/Munc18 (SM) proteins. The SNARE protein KNOLLE and the SM protein KEULE are required for membrane fusion at the cell plate. Due to the crucial function of KEULE, all Arabidopsis (Arabidopsis thaliana) keule mutants identified to date are seedling lethal. Here, we identified the Arabidopsis serrata4-1 (sea4-1) and sea4-2 mutants, which carry recessive, hypomorphic alleles of KEULE. Homozygous sea4-1 and sea4-2 plants are viable and fertile but have smaller rosettes and fewer leaves at bolting than the wild type. Their leaves are serrated, small, and wavy, with a complex venation pattern. The mutant leaves also develop necrotic patches and undergo premature senescence. RNA-seq revealed transcriptome changes likely leading to reduced cell wall integrity and an increase in the unfolded protein response. These findings shed light on the roles of KEULE in postembryonic development, particularly in the patterning of rosette leaves and leaf margins.

Genome-Wide Identification and Characterization of Lignin Synthesis Genes in Maize.

Lignin is a crucial substance in the formation of the secondary cell wall in plants. It is widely distributed in various plant tissues and plays a significant role in various biological processes. However, the number of copies, characteristics, and expression patterns of genes involved in lignin biosynthesis in maize are not fully understood. In this study, bioinformatic analysis and gene expression analysis were used to discover the lignin synthetic genes, and two representative maize inbred lines were used for stem strength phenotypic analysis and gene identification. Finally, 10 gene families harboring 117 related genes involved in the lignin synthesis pathway were retrieved in the maize genome. These genes have a high number of copies and are typically clustered on chromosomes. By examining the lignin content of stems and the expression patterns of stem-specific genes in two representative maize inbred lines, we identified three potential stem lodging resistance genes and their interactions with transcription factors. This study provides a foundation for further research on the regulation of lignin biosynthesis and maize lodging resistance genes.

OsWRKY12 negatively regulates the drought-stress tolerance and secondary cell wall biosynthesis by targeting different downstream transcription factor genes in rice.

With the increasing occurrence of global warming, drought is becoming a major constraint for plant growth and crop yield. Plant cell walls experience continuous changes during the growth, development, and in responding to stressful conditions. The plant WRKYs play pivotal roles in regulating the secondary cell wall (SCW) biosynthesis and helping plant defend against abiotic stresses. qRT-PCR evidence showed that OsWRKY12 was affected by drought and ABA treatments. Over-expression of OsWRKY12 decreased the drought tolerance of the rice transgenics at the germination stage and the seedling stage. The transcription levels of drought-stress-associated genes as well as those genes participating in the ABA biosynthesis and signaling were significantly different compared to the wild type (WT). Our results also showed that less lignin and cellulose were deposited in the OsWRKY12-overexpressors, and heterogenous expression of OsWRKY12 in atwrky12 could lower the increased lignin and cellulose contents, as well as the improved PEG-stress tolerance, to a similar level as the WT. qRT-PCR results indicated that the transcription levels of all the genes related to lignin and cellulose biosynthesis were significantly decreased in the rice transgenics than the WT. Further evidence from yeast one-hybrid assay and the dual-luciferase reporter system suggested that OsWRKY12 could bind to promoters of OsABI5 (the critical component of the ABA signaling pathway) and OsSWN3/OsSWN7 (the key positive regulators in the rice SCW thickening), and hence repressing their expression. In conclusion, OsWRKY12 mediates the crosstalk between SCW biosynthesis and plant stress tolerance by binding to the promoters of different downstream genes.

The conserved σD envelope stress response monitors multiple aspects of envelope integrity in corynebacteria.

The cell envelope fortifies bacterial cells against antibiotics and other insults. Species in the Mycobacteriales order have a complex envelope that includes an outer layer of mycolic acids called the mycomembrane (MM) and a cell wall composed of peptidoglycan and arabinogalactan. This envelope architecture is unique among bacteria and contributes significantly to the virulence of pathogenic Mycobacteriales like Mycobacterium tuberculosis. Characterization of pathways that govern envelope biogenesis in these organisms is therefore critical in understanding their biology and for identifying new antibiotic targets. To better understand MM biogenesis, we developed a cell sorting-based screen for mutants defective in the surface exposure of a porin normally embedded in the MM of the model organism Corynebacterium glutamicum. The results revealed a requirement for the conserved σD envelope stress response in porin export and identified MarP as the site-1 protease, respectively, that activate the response by cleaving the membrane-embedded anti-sigma factor. A reporter system revealed that the σD pathway responds to defects in mycolic acid and arabinogalactan biosynthesis, suggesting that the stress response has the unusual property of being induced by activating signals that arise from defects in the assembly of two distinct envelope layers. Our results thus provide new insights into how C. glutamicum and related bacteria monitor envelope integrity and suggest a potential role for members of the σD regulon in protein export to the MM.

The MAPK homolog, Smk1, promotes assembly of the glucan layer of the spore wall in S. cerevisiae.

Smk1 is a MAPK homolog in the yeast Saccharomyces cerevisiae that controls the postmeiotic program of spore wall assembly. During this program, haploid cells are surrounded by a layer of mannan and then a layer of glucan. These inner layers of the spore wall resemble the vegetative cell wall. Next, the outer layers consisting of chitin/chitosan and then dityrosine are assembled. The outer layers are spore-specific and provide protection against environmental stressors. Smk1 is required for the proper assembly of spore walls. However, the protective properties of the outer layers have limited our understanding of how Smk1 controls this morphogenetic program. Mutants lacking the chitin deacetylases, Cda1 and Cda2, form spores that lack the outer layers of the spore wall. In this study, cda1,2∆ cells were used to demonstrate that Smk1 promotes deposition of the glucan layer of the spore wall through the partially redundant glucan synthases Gsc2 and Fks3. Although Gsc2 is localized to sites of spore wall assembly in the wild type, it is mislocalized in the mother cell cytoplasm in the smk1∆ mutant. These findings suggest that Smk1 controls assembly of the spore wall by regulating the localization of Gsc2 during sporogenesis.

MicroRNA257 promotes secondary growth in hybrid poplar.

Populus, a significant fast-growing tree species with global afforestation and energy potential, holds considerable economic value. The abundant production of secondary xylem by trees, which serves as a vital resource for industrial purposes and human sustenance, necessitates the orchestration of various regulatory mechanisms, encompassing transcriptional regulators and microRNAs (miRNAs). Nevertheless, the investigation of microRNA-mediated regulation of poplar secondary growth remains limited. In this study, we successfully isolated a novel microRNA (Pag-miR257) from 84 K poplar and subsequently integrated it into the 35 S overexpression vector. The overexpression of Pag-miR257 resulted in notable increases in plant height, stem diameter, and fresh weight. Additionally, the overexpression of Pag-miR257 demonstrated a significant enhancement in net photosynthetic rate. The findings from the examination of cell wall autofluorescence indicated a substantial increase in both xylem area and the number of vessels in poplar plants overexpressing Pag-miR257. Furthermore, the cell wall of the Pag-miR257 overexpressing plants exhibited thickening as observed through transmission electron microscopy. Moreover, the Fourier Transforms Infrared (FTIR) analysis and phloroglucinol-HCl staining revealed an elevation in lignin content in Pag-miR257 overexpressing poplar plants. The findings of this study suggest that microRNA257 may play a role in the control of secondary growth in poplar stems, thereby potentially enhancing the development of wood engineering techniques for improved material and energy production.

Lysine 2-hydroxyisobutyrylation proteomics analyses reveal the regulatory mechanism of CaMYB61-CaAFR1 module in regulating stem development in Capsicum annuum L.

Plant stems constitute the most abundant renewable resource on earth. The function of lysine (K)-2-hydroxyisobutyrylation (Khib), a novel post-translational modification (PTM), has not yet been elucidated in plant stem development. Here, by assessing typical pepper genotypes with straight stem (SS) and prostrate stem (PS), we report the first large-scale proteomics analysis for protein Khib to date. Khib-modifications influenced central metabolic processes involved in stem development, such as glycolysis/gluconeogenesis and protein translation. The high Khib level regulated gene expression and protein accumulation associated with cell wall formation in the pepper stem. Specially, we found that CaMYB61 knockdown lines that exhibited prostrate stem phenotypes had high Khib levels. Most histone deacetylases (HDACs, e.g., switch-independent 3 associated polypeptide function related 1, AFR1) potentially function as the "erasing enzymes" involved in reversing Khib level. CaMYB61 positively regulated CaAFR1 expression to erase Khib and promote cellulose and hemicellulose accumulation in the stem. Therefore, we propose a bidirectional regulation hypothesis of "Khib modifications" and "Khib erasing" in stem development, and reveal a novel epigenetic regulatory network in which the CaMYB61-CaAFR1 molecular module participating in the regulation of Khib levels and biosynthesis of cellulose and hemicellulose for the first time.

Adaptation mechanism of three Impatiens species to different habitats based on stem morphology, lignin and MYB4 gene.

BACKGROUND: Impatiens is an important genus with rich species of garden plants, and its distribution is extremely extensive, which is reflected in its diverse ecological environment. However, the specific mechanisms of Impatiens' adaptation to various environments and the mechanism related to lignin remain unclear. RESULTS: Three representative Impatiens species,Impatiens chlorosepala (wet, low degree of lignification), Impatiens uliginosa (aquatic, moderate degree of lignification) and Impatiens rubrostriata (terrestrial, high degree of lignification), were selected and analyzed for their anatomical structures, lignin content and composition, and lignin-related gene expression. There are significant differences in anatomical parameters among the stems of three Impatiens species, and the anatomical structure is consistent with the determination results of lignin content. Furthermore, the thickness of the xylem and cell walls, as well as the ratio of cell wall thickness to stem diameter have a strong correlation with lignin content. The anatomical structure and degree of lignification in Impatiens can be attributed to the plant's growth environment, morphology, and growth rate. Our analysis of lignin-related genes revealed a negative correlation between the MYB4 gene and lignin content. The MYB4 gene may control the lignin synthesis in Impatiens by controlling the structural genes involved in the lignin synthesis pathway, such as HCT, C3H, and COMT. Nonetheless, the regulation pathway differs between species of Impatiens. CONCLUSIONS: This study demonstrated consistency between the stem anatomy of Impatiens and the results obtained from lignin content and composition analyses. It is speculated that MYB4 negatively regulates the lignin synthesis in the stems of three Impatiens species by regulating the expression of structural genes, and its regulation mechanism appears to vary across different Impatiens species. This study analyses the variations among different Impatiens plants in diverse habitats, and can guide further molecular investigations of lignin biosynthesis in Impatiens.

Dissecting the Ca2+ dependence of DesA1 function in Mycobacterium tuberculosis.

Mycobacterium tuberculosis (M. tb) has a complex cell wall, composed largely of mycolic acids, that are crucial to its structural maintenance. The M. tb desaturase A1 (DesA1) is an essential Ca2+-binding protein that catalyses a key step in mycolic acid biosynthesis. To investigate the structural and functional significance of Ca2+ binding, we introduced mutations at key residues in its Ca2+-binding ßγ-crystallin motif to generate DesA1F303A, E304Q, and F303A-E304Q. Complementation of a conditional ΔdesA1 strain of Mycobacterium smegmatis, with the Ca2+ non-binders F303A or F303A-E304Q, failed to rescue its growth phenotype; these complements also exhibited enhanced cell wall permeability. Our findings highlight the criticality of Ca2+ in DesA1 function, and its implicit role in the maintenance of mycobacterial cellular integrity.

Engineering of chimeric enzymes with expanded tolerance to ionic strength.

Antimicrobial resistance poses a significant global threat, reaching dangerously high levels as reported by the World Health Organization. The emergence and rapid spread of new resistance mechanisms, coupled with the absence of effective treatments in recent decades, have led to thousands of deaths annually from infections caused by drug-resistant microorganisms. Consequently, there is an urgent need for the development of new compounds capable of combating antibiotic-resistant bacteria. A promising class of molecules exhibiting potent bactericidal effects is peptidoglycan hydrolases. Previously, we cloned and characterized the biochemical properties of the M23 catalytic domain of the EnpA (EnpACD) protein from Enterococcus faecalis. Unlike other enzymes within the M23 family, EnpACD demonstrates broad specificity. However, its activity is constrained under low ionic strength conditions. In this study, we present the engineering of three chimeric enzymes comprising EnpACD fused with three distinct SH3b cell wall-binding domains. These chimeras exhibit enhanced tolerance to environmental conditions and sustained activity in bovine and human serum. Furthermore, our findings demonstrate that the addition of SH3b domains influences the activity of the chimeric enzymes, thereby expanding their potential applications in combating antimicrobial resistance.IMPORTANCEThese studies demonstrate that the addition of the SH3b-binding domain to the EnpACD results in generation of chimeras with a broader tolerance to ionic strength and pH values, enabling them to remain active over a wider range of conditions. Such approach offers a relatively straightforward method for obtaining antibacterial enzymes with tailored properties and emphasizes the potential for proteins' engineering with enhanced functionality, contributing to the ongoing efforts to address antimicrobial resistance effectively.

Hypothesis: evidence that the PRS gene products of Saccharomyces cerevisiae support both PRPP synthesis and maintenance of cell wall integrity.

The gene products of PRS1-PRS5 in Saccharomyces cerevisiae are responsible for the production of PRPP (5-phospho-D-ribosyl-α-1-pyrophosphate). However, it has been demonstrated that they are also involved in the cell wall integrity (CWI) signalling pathway as shown by protein-protein interactions (PPIs) with, for example Slt2, the MAP kinase of the CWI pathway. The following databases: SGD, BioGRID and Hit Predict, which collate PPIs from various research papers, have been scrutinized for evidence of PPIs between Prs1-Prs5 and components of the CWI pathway. The level of certainty in PPIs was verified by interaction scores available in the Hit Predict database revealing that well-documented interactions correspond with higher interaction scores and can be graded as high confidence interactions based on a score > 0.28, an annotation score ≥ 0.5 and a method-based high confidence score level of ≥ 0.485. Each of the Prs1-Prs5 polypeptides shows some degree of interaction with the CWI pathway. However, Prs5 has a vital role in the expression of FKS2 and Rlm1, previously only documented by reporter assay studies. This report emphasizes the importance of investigating interactions using more than one approach since every method has its limitations and the use of different methods, as described herein, provides complementary experimental and statistical data, thereby corroborating PPIs. Since the experimental data described so far are consistent with a link between PRPP synthetase and the CWI pathway, our aim was to demonstrate that these data are also supported by high-throughput bioinformatic analyses promoting our hypothesis that two of the five PRS-encoding genes contain information required for the maintenance of CWI by combining data from our targeted approach with relevant, unbiased data from high-throughput analyses.

A MYB-related transcription factor ZmMYBR29 is involved in grain filling.

BACKGROUND: The endosperm serves as the primary source of nutrients for maize (Zea mays L.) kernel embryo development and germination. Positioned at the base of the endosperm, the transfer cells (TCs) of the basal endosperm transfer layer (BETL) generate cell wall ingrowths, which enhance the connectivity between the maternal plant and the developing kernels. These TCs play a crucial role in nutrient transport and defense against pathogens. The molecular mechanism underlying BETL development in maize remains unraveled. RESULTS: This study demonstrated that the MYB-related transcription factor ZmMYBR29, exhibited specific expression in the basal cellularized endosperm, as evidenced by in situ hybridization analysis. Utilizing the CRISPR/Cas9 system, we successfully generated a loss-of-function homozygous zmmybr29 mutant, which presented with smaller kernel size. Observation of histological sections revealed abnormal development and disrupted morphology of the cell wall ingrowths in the BETL. The average grain filling rate decreased significantly by 26.7% in zmmybr29 mutant in comparison to the wild type, which impacted the dry matter accumulation within the kernels and ultimately led to a decrease in grain weight. Analysis of RNA-seq data revealed downregulated expression of genes associated with starch synthesis and carbohydrate metabolism in the mutant. Furthermore, transcriptomic profiling identified 23 genes that expressed specifically in BETL, and the majority of these genes exhibited altered expression patterns in zmmybr29 mutant. CONCLUSIONS: In summary, ZmMYBR29 encodes a MYB-related transcription factor that is expressed specifically in BETL, resulting in the downregulation of genes associated with kernel development. Furthermore, ZmMYBR29 influences kernels weight by affecting the grain filling rate, providing a new perspective for the complementation of the molecular regulatory network in maize endosperm development.