Feruloylated oligosaccharides (FOs) are bioactive conjugates that exhibit promising synergistic effects on gut microbiota modulation and intestinal barrier protection, yet their efficacy is highly structure-dependent. This study developed a synergistic strategy combining ionic liquid (IL, 1-butyl-3-methylimidazolium chloride) pretreatment with xylanase hydrolysis to selectively produce feruloylated arabinoxylobiose (FAX2) and feruloylated arabinoxylotriose (FAX3) from corn bran. The IL-pretreated FOs (FOsA) achieved a FAX3/FAX2 ratio of 4.86, which was 2.16-fold higher than that of the non-pretreated group (FOsB). Although in vitro assays confirmed that FOs were not directly utilized as a carbon source by Akkermansia muciniphila (A. muciniphila), both FOsA and FOsB significantly modulated its mucin-degrading enzyme activities (p < 0.05). Specifically, both fractions increased sialidase activity while reducing α-N-acetylglucosaminidase and β-galactosidase activities. Notably, FOsA, with a higher FAX3/FAX2 ratio, exhibited significantly stronger inhibition of exo-α-L-fucosidase activity than FOsB (p < 0.05). These findings demonstrate that FOs act as effective modulators of A. muciniphila enzymatic functions rather than metabolic substrates, and that their fine structural composition critically influences their regulatory potency on mucin-degrading enzymes, with potential implications for maintaining intestinal mucus barrier integrity.

The CRISPR/Cas9 system offers a powerful tool for gene editing to enhance rice productivity. In this study, we successfully edited eight RR-TZF genes in japonica rice Nipponbare using CRISPR-Cas9 technology, achieving a high editing efficiency of 73.8%. Sequencing revealed predominantly short insertions or deletions near the PAM sequence, along with multi-base deletions often flanked by identical bases. Off-target analysis identified 5 out of 31 predicted sites, suggesting the potential for off-target effects, which can be mitigated by designing gRNAs with more than three base mismatches. Notably, new mutations emerged in the progeny of several gene-edited mutants, indicating inheritable genetic mutagenicity. Phenotypic analysis of homozygous mutants revealed varied agronomic traits, even within the same gene, highlighting the complexity of gene-editing outcomes. These findings underscore the importance of backcrossing to minimize off-target and inheritable mutagenicity effects, ensuring more accurate trait evaluation. This study offers insights into CRISPR/Cas9 mechanisms and uncertain factors and may inform future strategies for rice improvement, prompting further research into CRISPR/Cas9’s precision and long-term impacts.

Verticillium is a small genus of ascomycete fungi containing 10 species. Several of these species are destructive phytopathogens that cause vascular diseases on diverse crops, including ornamentals, in different regions of the world. Traditional methods of differentiating the species based on morphological characteristics can lead to misidentification. Although rapid PCR detection assays are available, there remains a lack of universal and accurate methods for the identification of the 10 species. The aim of this research was to develop novel species-specific primers for the molecular detection of 10 Verticillium species. The primers were designed based on screening of species-specific genomic regions. Among the 10 primer sets tested, the detection limits ranged from approximately 0.1 to 1 ng of Verticillium DNA. The primer sets were further assessed on a collection of Verticillium strains from 10 species. Additionally, DNA extracted from two plant species or soil samples infested with eight different species was employed to further validate the utility of these primers. These species-specific primers facilitate the rapid identification of Verticillium species.

Recent advances in single-cell transcriptomics have greatly expanded our knowledge of plant development and cellular responses. However, analyzing fiber cell differentiation in plants, particularly in cotton, remains a complex challenge. A spatial transcriptomic map of ovule from -1 DPA, 0 DPA, and 1 DPA in cotton was successfully constructed, which helps to explain the important role of sucrose synthesis and lipid metabolism during early fiber development. Additionally, single-cell RNA sequencing (scRNA-seq) further highlighted the cellular heterogeneity and identified clusters of fiber developmental marker genes. Integration of spatial and scRNA-seq data unveiled key genes SVB and SVBL involved in fiber initiation, suggesting functional redundancy between them. These findings provide a detailed molecular landscape of cotton fiber development, offering valuable insights for enhancing lint yield.

Sugar beet (Beta vulgaris L.) is cultivated extensively worldwide as an important cash crop, and soil salinity is a critical factor influencing both its yield and sugar content. Consequently, enhancing the salt tolerance of sugar beet is of paramount importance. Arbuscular mycorrhizal (AM) fungi form symbiotic associations with approximately 80% of vascular plants, thereby improving the adaptability of host plants to adverse conditions. However, the mechanisms by which the AM symbiosis assists sugar beet in coping with salt stress remain poorly understood. To investigate the adaptation strategies employed by AM symbiotic sugar beet under salt stress, we examined physiological and transcriptomic changes in sugar beet seedlings subjected to various treatments, using the KWS1176 variety as the experimental material. The results indicated that AM symbiotic sugar beet demonstrated superior performance under salt stress, characterized by improved seedling growth, alterations in antioxidant enzyme activities, modifications in osmoregulatory substance levels, reduced Na+ uptake, and enhanced K+ influx within the root system. Notably, most of the differentially expressed genes were implicated in pathways related to reactive oxygen species scavenging, phenylpropanoid biosynthesis, and phytohormone signal transduction. Furthermore, pivotal genes identified through weighted gene co-expression network analysis were validated via reverse transcription-quantitative PCR, revealing that the salt tolerance of AM symbiotic sugar beet may be associated with its ionic homeostasis, antioxidant enzyme activities, and regulation of photosynthesis at both transcriptional and physiological levels.

Grain weight is a major determinant of wheat (Triticum aestivum L.) yield and is controlled by quantitative trait loci (QTLs) derived from natural variations. Here, we report the cloning of a major yield QTL in bread wheat that functions in the chlorophyll biosynthesis pathway. We found that the natural variation of CHELATASE SUBUNIT I-A (CHLI-A), encoding a magnesium chelatase subunit, enhances chlorophyll content and photosynthesis rate, leading to increased leaf area and grain weight. Increasing CHLI-A expression increases grain weight, indicating that photosynthesis is a limiting factor of wheat yield potential. The CHLI-A gene is absent in a portion of elite varieties, suggesting the potential of using this gene to improve grain yield in wheat.

Soil salinization is a threat to global agriculture. This study used whole-genome bisulfite sequencing and transcriptomics to explore epigenetic regulation in salt-tolerant Brassica napus (cv. Huayouza 62) under NaCl (salt), Na2CO3 (alkali), and combined salt-alkali stresses. All stresses induced genome-wide DNA hypermethylation (combined > alkali > salt). The Cn subgenome had significant methylation changes (most at CHH, then at CHG), with more differentially methylated regions in transposable elements than protein-coding genes. Hypermethylation, driven by DNA methyltransferases (e.g., DRM2), inhibited growth by repressing photosynthetic genes (RBCS-1A, RBCS-1B). B. napus adapted via targeted demethylation activated stress resistance (ROS scavenging under salt, redox buffering under alkali, and protein homeostasis under combined stress). 5-Azacytidine validation confirmed that methylation balances photosynthesis and stress response, clarifying the epigenetic network and providing strategies for crop stress resistance improvement.

In this study, the toxigenic characteristics of 14 strains of Microcystis were analyzed, and single nucleotide polymorphism (SNP) and insertion/deletion (InDel) loci in microcystin synthetase (mcy) gene clusters were screened. Based on SNP and InDel loci associated with the toxigenic characteristics, primers and TaqMan or Cycling fluorescent probes were designed to develop duplex real-time fluorescent quantitative PCR (FQ-PCR) assays. After evaluating specificity and sensitivity, these assays were applied to detect the toxigenic Microcystis genotypes in a shrimp pond where Microcystis blooms occurred. The results showed a total of 2155 SNP loci and 66 InDel loci were obtained, of which 12 SNP loci and 5 InDel loci were associated with the toxigenic characteristics. Three duplex real-time FQ-PCR assays were developed, each of which could quantify two genotypes of toxigenic Microcystis. These FQ-PCR assays were highly specific, and two Cycling assays were more sensitive than TaqMan assay. In the shrimp pond, six genotypes of toxigenic Microcystis were detected using the developed FQ-PCR assays, indicating that above genotyping assays have the potential for quantitative analysis of the toxigenic Microcystis genotypes in natural water.

Florets are the basic structural units of spikelets, and their morphogenesis determines the yield and quality of rice grains. However, whether and how pseudouridine-5′-phosphate glycosidase participates in rice spikelet development remains an open question. In this study, we identified a novel gene, OsPPG, which encodes a peroxisome-localized pseudouridine-5′-phosphate glycosidase and regulates the development of rice spikelets. osppg mutants exhibited abnormal lemma, palea, stamens, and pistils; male sterility; shorter panicles; and reduced plant height. OsPPG was found to regulate several OsMADS genes, thereby affecting the morphogenesis of rice spikelets. Furthermore, metabolomics revealed that the OsPPG gene was involved in the decomposition of pseudouridine via the pyrimidine metabolism pathway and may affect the jasmonic acid signaling pathway. These results suggest that OsPPG is a key regulator of rice spikelet development.

Molecular markers play a crucial role in marker-assisted breeding and varietal identification. However, the application of insertion/deletion markers (InDels) in grapevines has been limited by the low throughput and separability of gel electrophoresis. To developed effective InDel markers for grapevines, this study reports a novel, effective and high-throughput pipeline for InDel marker development and identification. After rigorous filtering, 11 polymorphic multi-allelic InDel markers were selected. These markers were then used to perform genetic identification of 123 elite grape cultivars using agarose gel electrophoresis and next-generation sequencing (NGS). The polymorphism rate of the InDel markers identified by gels was 37.92%, while the NGS-based results demonstrated a higher polymorphism rate of 61.12%. Finally, the NGS-based fingerprints successfully distinguished 122 grape varieties (99.19%), surpassing the gels, which could distinguish 116 grape varieties (94.31%). Specifically, we constructed phylogenetic trees based on the genotyping results from both gels and NGS. The population structure revealed by the NGS-based markers displayed three primary clusters, consisting of the patterns of the evolutionary divergence and geographical origin of the grapevines. Our work provides an efficient workflow for multi-allelic InDel marker development and practical tools for the genetic discrimination of grape cultivars.

Cauliflower is a distinct subspecies of the Brassica oleracea plants due to its specialized and edible floral organ. Cauliflower curd is composed of enlarged inflorescence meristems that developed by a series of precise molecular regulations. Based solely on the curd solidity, cauliflower is generally classified into two groups (compact-curd and loose-curd), where curd branch length acts as a crucial parameter to determine the curd morphological difference. Herein, to understand the genetic basis of curd branch development, we utilized a total of 298 inbred lines representing two groups of cauliflower to comprehensively investigate the causal genes and regulatory mechanisms. Phylogenetic and population structure analyses revealed that two subgroups could be further categorized into the compact-curd and the loose-curd groups, respectively. Integrating the genotype and phenotype data, we conducted a genome-wide association study for the length of the outermost branch (LOB) and secondary branch (LSB) of the curd. Sixty-four significant loci were identified that are highly associated with curd branch development. Evidence from genome-wide selective sweep analysis (FST and XP-EHH) narrowed down the major signal on chromosome 8 into an approximately 79 kb region which encodes eleven protein-coding genes. After further analysis of haplotypes, transcriptome profiling, and gene expression validation, we finally inferred that BOB08G028680, as a homologous counterpart of AtARR9, might be the causal gene for simultaneously regulating LOB and LSB traits in cauliflower. This result provides valuable information for improving curd solidity in future cauliflower breeding.

MicroRNAs (miRNAs) are versatile regulators of gene expression at both the transcription and post-transcription levels. The microRNA miR396 plays vital roles in growth, development, and resistance to abiotic stresses in many plant species. However, the roles and functions of miR396 in soybeans are not well understood. Here, we report that Gm-miR396a influences soybean development and salinity tolerance. We found that soybean miR396a was responsive to salt stress. Gm-miR396a gene-edited lines (miR396a-GEs), created using CRISPR/Cas9, exhibited more branches, higher grain yields, and greater salinity tolerance than control plants. The transcripts in lines with altered abundance of miR396a-GE were significantly enriched for biological processes related to hormone regulation. Overexpression of the Gm-miR396a precursor (pre-miR396a-OE) resulted in developmental deficiencies including dwarfness, abnormal inflorescences and flowers, smaller and fewer seeds, and small leaves with larger and more numerous stomata. Transcriptome analysis indicated photosynthesis-related genes were downregulated in pre-miR396a-OE plants. These results contribute valuable insights into the function of Gm-miR396a in soybeans and hold promise for enhancing soybean yield and salinity tolerance through germplasm innovation.

Florets are the basic structural units of spikelets, and their morphogenesis determines the yield and quality of rice grains. However, whether and how pseudouridine-5 0-phosphate glycosidase participates in rice spikelet development remains an open question. In this study, we identified a novel gene, OsPPG , which encodes a peroxisome-localized pseudouridine-5 0-phosphate glycosidase and regulates the development of rice spikelets. osppg mutants exhibited abnormal sterile lemma, lemma, palea, lodicule, stamens, and pistils; male sterility; shorter panicles; and reduced plant height. OsPPG was found to regulate several OsMADS genes, thereby affecting the morphogenesis of rice spikelets. Furthermore, metabolomics revealed that the OsPPG gene was involved in the decomposition of pseudouridine via the pyrimidine metabolism pathway and may affect the jasmonic acid signaling pathway. These results suggest that OsPPG is a key regulator of rice spikelet development. (c) 2024 Crop Science Society of China and Institute of Crop Science, CAAS. Production and hosting by Elsevier B.V. on behalf of KeAi Communications Co., Ltd. This is an open access article under the CC BY-NCND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).

Cauliflower (Brassica oleracea L. var. botrytis) is a distinctive vegetable that supplies a nutrient-rich edible inflorescence meristem for the human diet. However, the genomic bases of its selective breeding have not been studied extensively. Herein, we present a high-quality reference genome assembly C-8 (V2) and a comprehensive genomic variation map consisting of 971 diverse accessions of cauliflower and its relatives. Genomic selection analysis and deep-mined divergences were used to explore a stepwise domestication process for cauliflower that initially evolved from broccoli (Curd-emergence and Curd-improvement), revealing that three MADS-box genes, CAULIFLOWER1 (CAL1), CAL2 and FRUITFULL (FUL2), could have essential roles during curd formation. Genome-wide association studies identified nine loci significantly associated with morphological and biological characters and demonstrated that a zinc-finger protein (BOB06G135460) positively regulates stem height in cauliflower. This study offers valuable genomic resources for better understanding the genetic bases of curd biogenesis and florescent development in crops. A high-quality reference genome assembly of cauliflower C-8 (V2) and genomic analyses of 971 diverse accessions and their relatives reveal the stepwise domestication and the genetic mechanism of curd biogenesis.

Genetically modified (GM) soybeans provide a huge amount of food for human consumption and animal feed. However, the possibility of unexpected effects of transgenesis has increased food safety concerns. High-throughput sequencing profiling provides a potential approach to directly evaluate unintended effects caused by foreign genes. In this study, we performed transcriptomic analyses to evaluate differentially expressed genes (DEGs) in individual soybean tissues, including cotyledon (C), germ (G), hypocotyl (H), and radicle (R), instead of using the whole seed, from four GM and three non-GM soybean lines. A total of 3,351 DEGs were identified among the three non-GM soybean lines. When the GM lines were compared with their non-GM parents, 1,836 to 4,551 DEGs were identified. Furthermore, Gene Ontology (GO) analysis of the DEGs showed more abundant categories of GO items (199) among non-GM lines than between GM lines and the non-GM natural varieties (166). Results of Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis showed that most KEGG pathways were the same for the two types of comparisons. The study successfully employed RNA sequencing to assess the differences in gene expression among four tissues of seven soybean varieties, and the results suggest that transgenes do not induce massive transcriptomic alterations in transgenic soybeans compared with those that exist among natural varieties. This work offers empirical evidence to investigate the genomic-level disparities induced by genetic modification in soybeans, specifically focusing on seed tissues.

Background Bud sport mutation occurs frequently in fruit plants and acts as an important approach for grapevine improvement and breeding. ‘Jinzao Wuhe’ is a bud sport of the elite cultivar ‘Himord Seedless’ with obviously enlarged organs and berries. To date, the molecular mechanisms underlying berry enlargement caused by bud sport in grapevines remain unclear. Methods Whole genome resequencing (WGRS) was performed for two pairs of bud sports and their maternal plants with similar phenotype to identify SNPs, InDels and structural variations (SVs) as well as related genes. Furthermore, transcriptomic sequencing at different developmental stages and weighted gene co-expression network analysis (WGCNA) for ‘Jinzao Wuhe’ and its maternal plant ‘Himord Seedless’ were carried out to identify the differentially expressed genes (DEGs), which were subsequently analyzed for Gene Ontology (GO) and function annotation. Results In two pairs of enlarged berry bud sports, a total of 1,334 SNPs, 272 InDels and 74 SVs, corresponding to 1,022 target genes related to symbiotic microorganisms, cell death and other processes were identified. Meanwhile, 1,149 DEGs associated with cell wall modification, stress-response and cell killing might be responsible for the phenotypic variation were also determined. As a result, 42 DEGs between ‘Himord Seedless’ and ‘Jinzao Wuhe’ harboring genetic variations were further investigated, including pectin esterase, cellulase A, cytochromes P450 (CYP), UDP-glycosyltransferase (UGT), zinc finger protein, auxin response factor (ARF), NAC transcription factor (TF), protein kinase, etc . These candidate genes offer important clues for a better understanding of developmental regulations of berry enlargement in grapevine. Conclusion Our results provide candidate genes and valuable information for dissecting the underlying mechanisms of berry development and contribute to future improvement of grapevine cultivars.

The lithospheric microbiome plays a vital role in global biogeochemical cycling, yet their mutual modulation mechanisms remain largely uncharted. Petroleum reservoirs are important lithosphere ecosystems that provide desirable resources for understanding microbial roles in element cycling. However, the strategy and mechanism of modulating indigenous microbial communities for the optimization of community structures and functions are underexplored, despite its significance in energy recovery and environmental remediation. Here we proposed a novel selective stimulation of indigenous functional microbes by driving nitrogen and sulfur cycling in petroleum reservoirs using injections of an exogenous heterocycle-degrading strain of Pseudomonas. We defined such bacteria capable of removing and releasing organically bound sulfur and nitrogen from heterocycles as “bioredox triggers”. High-throughput 16S rRNA amplicon sequencing, metagenomic, and gene transcription-level analyses of extensive production water and sandstone core samples spanning the whole oil production process clarified the microbiome dynamics following the intervention. These efforts demonstrated the feasibility of in situ N/S element release and electron acceptor generation during heterocycle degradation, shifting microbiome structures and functions and increasing phylogenetic diversity and genera engaged in sulfur and nitrogen cycling, such as Desulfovibrio, Shewanella, and Sulfurospirillum. The metabolic potentials of sulfur- and nitrogen-cycling processes, particularly dissimilatory sulfate reduction and dissimilatory nitrate reduction, were elevated in reservoir microbiomes. The relative expression of genes involved in sulfate reduction (dsrA, dsrB) and nitrate reduction (napA) was upregulated by 85, 28, and 22 folds, respectively. Field trials showed significant improvements in oil properties, with a decline in asphaltenes and aromatics, hetero-element contents, and viscosity, hence facilitating the effective exploitation of heavy oil. The interactions between microbiomes and element cycling elucidated in this study will contribute to a better understanding of microbial metabolic involvement in, and response to, biogeochemical processes in the lithosphere. The presented findings demonstrated the immense potential of our microbial modulation strategy for green and enhanced heavy oil recovery.

Background Plant chloroplast DNA (cpDNA) typically has a circular structure, including a large single-copy region (LSC), a small single-copy region (SSC) and two inverted repeats (IR1 and IR2). The organization of these four elementary regions LSC-IR1-SSC-IR2 is highly conserved across all plant cpDNAs. Very few structural variations (SVs) occurring at the elementary-region level have been reported. Results In the present study, we assembled the full-length cpDNA of Dongxiang wild rice line 159 (DXWR159). Using the long PacBio subreads, we discovered a large inversion of SSC and a large duplication of IR in DXWR159 cpDNAs. Significantly, we reported for the first time forward and reverse SSCs of cpDNAs in similar proportions and named the frequent inversion of a whole SSC as SSC switching. Conclusions Our study helps researchers to correctly assemble the chloroplast genomes. Our recombination model explained the formation of large SVs in cpDNAs and provided insights into a novel scientific question that if there are common mechanisms in the formation or translocation of all kinds of transposon-like elements (TLEs). We propose that: (1) large inversion is the most accepted mutation type of SVs in cpDNAs; (2) SSC switching ubiquitous occurs in plant cpDNAs; and (3) further investigation of molecular mechanism underlying SSC switching may reveal new driving forces for large SVs.

Abstract Background Berry size and early-ripening are considered as crucial traits for grape production and breeding. However, the underlying molecular mechanisms regulating berry size and early-ripening caused by bud sport in grapes remain unknown. Results In this study, whole-genome resequencing was performed for the cultivars ‘Himord Seedless’, ‘Thompson Seedless’ and their larger berries and early-ripening bud sports, ‘Jinzao Wuhe’ and ‘Dawuhebai’. Consequently, 1,548 SNPs, 379 InDels and 481 SVs, corresponding to 1,440 target genes, were identified in both bud sports compared to their maternal plants. Transcriptome analysis for ‘Jinzao Wuhe’/‘Himord Seedless’ at four time-points, including full-flowering, berry expansion, véraison and maturity stages, generated 2,984 differential expression genes (DEGs). Of these, 121 DEGs comprise genetic variations which might play potential roles in controlling berry growth and maturity development. Furthermore, weighted gene co-expression network analysis (WGCNA) was carried out, which revealed 48 candidate genes with previously discovered SNPs, InDels and SVs, including MADS, NAC, WRKY, UDP-glycosyltransferases, cytochromes P450, and genes involved in cell wall modification, ethylene synthesis and plant resistance. Finally, a conceivable regulatory network was constructed by integrating entire clues about the function of target genes. Conclusions Combined with the whole-genome resequencing and RNA-seq, DEGs with genetic variations associated with early ripening and berry size were identified in ‘Himord Seedless’ and its bud variant ‘Jinzao Wuhe’. Our results gave new insights into the field of bud mutation and contributed to future grape breeding and genetic improvement.

As one of the largest transcription factor family, basic helix-loop-helix (bHLH) transcription factor family plays an important role in plant metabolism, physiology and growth. Berry color is one of the important factors that determine grape quality. However, the bHLH transcription factor family’s function in anthocyanin synthesis of grape berry has not been studied systematically. We identified 115 bHLH transcription factors in grape genome and phylogenetic analysis indicated that bHLH family could be classified into 25 subfamilies. First, we screened six candidate genes by bioinformatics analysis and expression analysis. We found one of the candidate genes VdbHLH037 belonged to III (f) subfamily and interacted with genes related to anthocyanin synthesis through phylogenetic analysis and interaction network prediction. Therefore, we speculated that VdbHLH037 participated in the anthocyanin synthesis process. To confirm this, we transiently expressed VdbHLH037 in grape and Arabidopsis transformation. Compared with the control, transgenic materials can accumulate more anthocyanins. These results provide a good base to study the function of the VdbHLH family in anthocyanin synthesis of grape berry.