Concordant Gene Expression and Alternative Splicing Regulation under Abiotic Stresses in Arabidopsis.

The current investigation endeavors to identify differentially expressed alternatively spliced (DAS) genes that exhibit concordant expression with splicing factors (SFs) under diverse multifactorial abiotic stress combinations in Arabidopsis seedlings. SFs serve as the post-transcriptional mechanism governing the spatiotemporal dynamics of gene expression. The different stresses encompass variations in salt concentration, heat, intensive light, and their combinations. Clusters demonstrating consistent expression profiles were surveyed to pinpoint DAS/SF gene pairs exhibiting concordant expression. Through rigorous selection criteria, which incorporate alignment with documented gene functionalities and expression patterns observed in this study, four members of the serine/arginine-rich (SR) gene family were delineated as SFs concordantly expressed with six DAS genes. These regulated SF genes encompass cactin, SR1-like, SR30, and SC35-like. The identified concordantly expressed DAS genes encode diverse proteins such as the 26.5 kDa heat shock protein, chaperone protein DnaJ, potassium channel GORK, calcium-binding EF hand family protein, DEAD-box RNA helicase, and 1-aminocyclopropane-1-carboxylate synthase 6. Among the concordantly expressed DAS/SF gene pairs, SR30/DEAD-box RNA helicase, and SC35-like/1-aminocyclopropane-1-carboxylate synthase 6 emerge as promising candidates, necessitating further examinations to ascertain whether these SFs orchestrate splicing of the respective DAS genes. This study contributes to a deeper comprehension of the varied responses of the splicing machinery to abiotic stresses. Leveraging these DAS/SF associations shows promise for elucidating avenues for augmenting breeding programs aimed at fortifying cultivated plants against heat and intensive light stresses.

Exploration of genes encoding KEGG pathway enzymes in rhizospheric microbiome of the wild plant Abutilon fruticosum.

The operative mechanisms and advantageous synergies existing between the rhizobiome and the wild plant species Abutilon fruticosum were studied. Within the purview of this scientific study, the reservoir of genes in the rhizobiome, encoding the most highly enriched enzymes, was dominantly constituted by members of phylum Thaumarchaeota within the archaeal kingdom, phylum Proteobacteria within the bacterial kingdom, and the phylum Streptophyta within the eukaryotic kingdom. The ensemble of enzymes encoded through plant exudation exhibited affiliations with 15 crosstalking KEGG (Kyoto Encyclopaedia of Genes and Genomes) pathways. The ultimate goal underlying root exudation, as surmised from the present investigation, was the biosynthesis of saccharides, amino acids, and nucleic acids, which are imperative for the sustenance, propagation, or reproduction of microbial consortia. The symbiotic companionship existing between the wild plant and its associated rhizobiome amplifies the resilience of the microbial community against adverse abiotic stresses, achieved through the orchestration of ABA (abscisic acid) signaling and its cascading downstream effects. Emergent from the process of exudation are pivotal bioactive compounds including ATP, D-ribose, pyruvate, glucose, glutamine, and thiamine diphosphate. In conclusion, we hypothesize that future efforts to enhance the growth and productivity of commercially important crop plants under both favorable and unfavorable environmental conditions may focus on manipulating plant rhizobiomes.

Metabolic analysis of the CAZy class glycosyltransferases in rhizospheric soil fungiome of the plant species Moringa oleifera.

The target of the present work is to study the most abundant carbohydrate-active enzymes (CAZymes) of glycosyltransferase (GT) class, which are encoded by fungiome genes present in the rhizospheric soil of the plant species Moringa oleifera. The datasets of this CAZy class were recovered using metagenomic whole shotgun genome sequencing approach, and the resultant CAZymes were searched against the KEGG pathway database to identify function. High emphasis was given to the two GT families, GT4 and GT2, which were the highest within GT class in the number and abundance of gene queries in this soil compartment. These two GT families harbor CAZymes playing crucial roles in cell membrane and cell wall processes. These CAZymes are responsible for synthesizing essential structural components such as cellulose and chitin, which contribute to the integrity of cell walls in plants and fungi. The CAZyme beta-1,3-glucan synthase of GT2 family accumulates 1,3-ß-glucan, which provides elasticity as well as tensile strength to the fungal cell wall. Other GT CAZymes contribute to the biosynthesis of several compounds crucial for cell membrane and wall integrity, including lipopolysaccharide, e.g., lipopolysaccharide N-acetylglucosaminyltransferase, cell wall teichoic acid, e.g., alpha-glucosyltransferase, and cellulose, e.g., cellulose synthase. These compounds also play pivotal roles in ion homeostasis, organic carbon mineralization, and osmoprotection against abiotic stresses in plants. This study emphasizes the major roles of these two CAZy GT families in connecting the structure and function of cell membranes and cell walls of fungal and plant cells. The study also sheds light on the potential occurrence of tripartite symbiotic relationships involving the plant, rhizospheric bacteriome, and fungiome via the action of CAZymes of GT4 and GT2 families. These findings provide valuable insights towards the generation of innovative agricultural practices to enhance the performance of crop plants in the future.

Assessing the probiotic potential, antioxidant, and antibacterial activities of oat and soy milk fermented with Lactiplantibacillus plantarum strains isolated from Tibetan Kefir.

Sufficient intake of probiotics has been shown to help in the digestion, protect the body against pathogenic microorganisms and boost the immune system. Recently, due to high prevalence of milk allergies and lactose intolerance in population, the non-dairy based probiotic alternative are becoming increasing popular. In this context, the oat milk and soya milk-based fermented products can be an ideal alternative for the development of Lactic acid bacteria bacteria based probiotics. These bacteria can not only improve the product's flavor and bioavailability but also increases its antibacterial and antioxidant capabilities due to fermentation process. The purpose of the resent work was to assess the antioxidant and probiotic properties of oat and soy milk that had been fermented with three different strains of Lactiplantibacillus plantarum (L. plantarum) including L. plantarum 12-3, L. plantarum K25, and L. plantarum YW11 isolated from Tibetan Kefir. Different validated assays were used to evaluate the probiotic properties, adhesion and survival in the digestive system (stomach, acid and bile salts resistance), antioxidant and antimicrobial activities and safety (ABTS and DPPH scavenging assays) of these strains. Results of the study showed that soya milk and oat milk fermented with L. plantarum strains possess promising probiotic, antibacterial and antioxidant properties. These results can be helpful to produce dairy-free probiotic replacements, which are beneficial for those who are unable to consume dairy products due to dietary or allergic restrictions.

Abundant resistome determinants in rhizosphere soil of the wild plant Abutilon fruticosum.

A metagenomic whole genome shotgun sequencing approach was used for rhizospheric soil micribiome of the wild plant Abutilon fruticosum in order to detect antibiotic resistance genes (ARGs) along with their antibiotic resistance mechanisms and to detect potential risk of these ARGs to human health upon transfer to clinical isolates. The study emphasized the potential risk to human health of such human pathogenic or commensal bacteria, being transferred via food chain or horizontally transferred to human clinical isolates. The top highly abundant rhizospheric soil non-redundant ARGs that are prevalent in bacterial human pathogens or colonizers (commensal) included mtrA, soxR, vanRO, golS, rbpA, kdpE, rpoB2, arr-1, efrA and ileS genes. Human pathogenic/colonizer bacteria existing in this soil rhizosphere included members of genera Mycobacterium, Vibrio, Klebsiella, Stenotrophomonas, Pseudomonas, Nocardia, Salmonella, Escherichia, Citrobacter, Serratia, Shigella, Cronobacter and Bifidobacterium. These bacteria belong to phyla Actinobacteria and Proteobacteria. The most highly abundant resistance mechanisms included antibiotic efflux pump, antibiotic target alteration, antibiotic target protection and antibiotic inactivation. antimicrobial resistance (AMR) families of the resistance mechanism of antibiotic efflux pump included resistance-nodulation-cell division (RND) antibiotic efflux pump (for mtrA, soxR and golS genes), major facilitator superfamily (MFS) antibiotic efflux pump (for soxR gene), the two-component regulatory kdpDE system (for kdpE gene) and ATP-binding cassette (ABC) antibiotic efflux pump (for efrA gene). AMR families of the resistance mechanism of antibiotic target alteration included glycopeptide resistance gene cluster (for vanRO gene), rifamycin-resistant beta-subunit of RNA polymerase (for rpoB2 gene) and antibiotic-resistant isoleucyl-tRNA synthetase (for ileS gene). AMR families of the resistance mechanism of antibiotic target protection included bacterial RNA polymerase-binding protein (for RbpA gene), while those of the resistance mechanism of antibiotic inactivation included rifampin ADP-ribosyltransferase (for arr-1 gene). Better agricultural and food transport practices are required especially for edible plant parts or those used in folkloric medicine.

Functional annotation of rhizospheric phageome of the wild plant species Moringa oleifera.

Introduction: The study aims to describe phageome of soil rhizosphere of M.oleifera in terms of the genes encoding CAZymes and other KEGG enzymes. Methods: Genes of the rhizospheric virome of the wild plant species Moringa oleifera were investigated for their ability to encode useful CAZymes and other KEGG (Kyoto Encyclopedia of Genes and Genomes) enzymes and to resist antibiotic resistance genes (ARGs) in the soil. Results: Abundance of these genes was higher in the rhizospheric microbiome than in the bulk soil. Detected viral families include the plant viral family Potyviridae as well as the tailed bacteriophages of class Caudoviricetes that are mainly associated with bacterial genera Pseudomonas, Streptomyces and Mycobacterium. Viral CAZymes in this soil mainly belong to glycoside hydrolase (GH) families GH43 and GH23. Some of these CAZymes participate in a KEGG pathway with actions included debranching and degradation of hemicellulose. Other actions include biosynthesizing biopolymer of the bacterial cell wall and the layered cell wall structure of peptidoglycan. Other CAZymes promote plant physiological activities such as cell-cell recognition, embryogenesis and programmed cell death (PCD). Enzymes of other pathways help reduce the level of soil H2O2 and participate in the biosynthesis of glycine, malate, isoprenoids, as well as isoprene that protects plant from heat stress. Other enzymes act in promoting both the permeability of bacterial peroxisome membrane and carbon fixation in plants. Some enzymes participate in a balanced supply of dNTPs, successful DNA replication and mismatch repair during bacterial cell division. They also catalyze the release of signal peptides from bacterial membrane prolipoproteins. Phages with the most highly abundant antibiotic resistance genes (ARGs) transduce species of bacterial genera Pseudomonas, Streptomyces, and Mycobacterium. Abundant mechanisms of antibiotic resistance in the rhizosphere include "antibiotic efflux pump" for ARGs soxR, OleC, and MuxB, "antibiotic target alteration" for parY mutant, and "antibiotic inactivation" for arr-1. Discussion: These ARGs can act synergistically to inhibit several antibiotics including tetracycline, penam, cephalosporin, rifamycins, aminocoumarin, and oleandomycin. The study highlighted the issue of horizontal transfer of ARGs to clinical isolates and human gut microbiome.

Antibiotic sensitivity and resistance of gastrointestinal microflora isolated from preterm neonates.

Aim: This study aimed to isolate and identify common bacteria from 19 preterm neonates who spent their first weeks in the neonatal intensive care unit. Materials & methods: Stool samples were collected, and bacteria were isolated and purified from the samples. The isolated bacterial species were tested for antibiotic susceptibility or resistance. Results: Three common species were found in 15 stool samples: Enterobacter cloacae, Enterococcus fecalis and Klebsiella pneumoniae. Minimum inhibitory concentrations determined using antibiotic susceptibility testing showed that the minimum level of isolates was affected by the most commonly used antibiotics, with significant resistance to some of the tested antibiotics. Conclusion: The development of beneficial normal flora in preterm neonates plays a vital role in their health and well-being.

Overlapping roles of spliceosomal components SF3B1 and PHF5A in rice splicing regulation.

The SF3B complex, a multiprotein component of the U2 snRNP of the spliceosome, plays a crucial role in recognizing branch point sequence and facilitates spliceosome assembly and activation. Several chemicals that bind SF3B1 and PHF5A subunits of the SF3B complex inhibit splicing. We recently generated a splicing inhibitor-resistant SF3B1 mutant named SF3B1 GEX1A RESISTANT 4 (SGR4) using CRISPR-mediated directed evolution, whereas splicing inhibitor-resistant mutant of PHF5A (Overexpression-PHF5A GEX1A Resistance, OGR) was generated by expressing an engineered version PHF5A-Y36C. Global analysis of splicing in wild type and these two mutants revealed the role of SF3B1 and PHF5A in splicing regulation. This analysis uncovered a set of genes whose intron retention is regulated by both proteins. Further analysis of these retained introns revealed that they are shorter, have a higher GC content, and contain shorter and weaker polypyrimidine tracts. Furthermore, splicing inhibition increased seedlings sensitivity to salt stress, consistent with emerging roles of splicing regulation in stress responses. In summary, we uncovered the functions of two members of the plant branch point recognition complex. The novel strategies described here should be broadly applicable in elucidating functions of splicing regulators, especially in studying the functions of redundant paralogs in plants.

CRISPR base editors: genome editing without double-stranded breaks.

The CRISPR (clustered regularly interspaced short palindromic repeat)/Cas9 adaptive immunity system has been harnessed for genome editing applications across eukaryotic species, but major drawbacks, such as the inefficiency of precise base editing and off-target activities, remain. A catalytically inactive Cas9 variant (dead Cas9, dCas9) has been fused to diverse functional domains for targeting genetic and epigenetic modifications, including base editing, to specific DNA sequences. As base editing does not require the generation of double-strand breaks, dCas9 and Cas9 nickase have been used to target deaminase domains to edit specific loci. Adenine and cytidine deaminases convert their respective nucleotides into other DNA bases, thereby offering many possibilities for DNA editing. Such base-editing enzymes hold great promise for applications in basic biology, trait development in crops, and treatment of genetic diseases. Here, we discuss recent advances in precise gene editing using different platforms as well as their potential applications in basic biology and biotechnology.

Herboxidiene triggers splicing repression and abiotic stress responses in plants.

BACKGROUND: Constitutive and alternative splicing of pre-mRNAs from multiexonic genes controls the diversity of the proteome; these precisely regulated processes also fine-tune responses to cues related to growth, development, and stresses. Small-molecule inhibitors that perturb splicing provide invaluable tools for use as chemical probes to uncover the molecular underpinnings of splicing regulation and as potential anticancer compounds. RESULTS: Here, we show that herboxidiene (GEX1A) inhibits both constitutive and alternative splicing. Moreover, GEX1A activates genome-wide transcriptional patterns involved in abiotic stress responses in plants. GEX1A treatment -activated ABA-inducible promoters, and led to stomatal closure. Interestingly, GEX1A and pladienolide B (PB) elicited similar cellular changes, including alterations in the patterns of transcription and splicing, suggesting that these compounds might target the same spliceosome complex in plant cells. CONCLUSIONS: Our study establishes GEX1A as a potent splicing inhibitor in plants that can be used to probe the assembly, dynamics, and molecular functions of the spliceosome and to study the interplay between splicing stress and abiotic stresses, as well as having potential biotechnological applications.

Pre-mRNA splicing repression triggers abiotic stress signaling in plants.

Alternative splicing (AS) of precursor RNAs enhances transcriptome plasticity and proteome diversity in response to diverse growth and stress cues. Recent work has shown that AS is pervasive across plant species, with more than 60% of intron-containing genes producing different isoforms. Mammalian cell-based assays have discovered various inhibitors of AS. Here, we show that the macrolide pladienolide B (PB) inhibits constitutive splicing and AS in plants. Also, our RNA sequencing (RNA-seq) data revealed that PB mimics abiotic stress signals including salt, drought and abscisic acid (ABA). PB activates the abiotic stress- and ABA-responsive reporters RD29A::LUC and MAPKKK18::uidA in Arabidopsis thaliana and mimics the effects of ABA on stomatal aperture. Genome-wide analysis of AS by RNA-seq revealed that PB perturbs the splicing machinery and leads to a striking increase in intron retention and a reduction in other forms of AS. Interestingly, PB treatment activates the ABA signaling pathway by inhibiting the splicing of clade A PP2C phosphatases while still maintaining to some extent the splicing of ABA-activated SnRK2 kinases. Taken together, our data establish PB as an inhibitor and modulator of splicing and a mimic of abiotic stress signals in plants. Thus, PB reveals the molecular underpinnings of the interplay between stress responses, ABA signaling and post-transcriptional regulation in plants.

Transcription activator-like effector nucleases mediated metabolic engineering for enhanced fatty acids production in Saccharomyces cerevisiae.

Targeted engineering of microbial genomes holds much promise for diverse biotechnological applications. Transcription activator-like effector nucleases (TALENs) and clustered regularly interspaced short palindromic repeats/Cas9 systems are capable of efficiently editing microbial genomes, including that of Saccharomyces cerevisiae. Here, we demonstrate the use of TALENs to edit the genome of S. cerevisiae with the aim of inducing the overproduction of fatty acids. Heterodimeric TALENs were designed to simultaneously edit the FAA1 and FAA4 genes encoding acyl-CoA synthetases in S. cerevisiae. Functional yeast double knockouts generated using these TALENs over-produce large amounts of free fatty acids into the cell. This study demonstrates the use of TALENs for targeted engineering of yeast and demonstrates that this technology can be used to stimulate the enhanced production of free fatty acids, which are potential substrates for biofuel production. This proof-of-principle study extends the utility of TALENs as excellent genome editing tools and highlights their potential use for metabolic engineering of yeast and other organisms, such as microalgae and plants, for biofuel production.