Mutant Phenotype Research Articles

Maize DLR1/NHX7 Is Required for Root Development Under Potassium Deficiency.

Root System Architecture (RSA) is a crucial plant trait that governs a plant's ability to absorb water and nutrients. In this study, we describe a mutant with nutrient-dependent defects in root development, affecting both the primary root and lateral roots (LRs). This mutant, identified through a screen for defects in LR development, has been designated dlr1-1. The dlr1-1 mutant exhibits impaired LR emergence rather than defects in the LR primordium (LRP) formation, particularly under potassium (K+)-deprivation conditions. This impairment likely stems from inhibited cell proliferation caused by the dlr1-1 mutation. K+ deprivation specifically leads to the accumulation of salicylic acid (SA) in the dlr1-1 mutant, consistent with the upregulation of SA biosynthesis genes. Moreover, exogenous application of SA to wild-type plants (B73) mimics the dlr1-1 phenotype. Conversely, treatment of the dlr1-1 mutant with 2-aminoindane-2-phosphonic acid, an SA biosynthesis inhibitor, partially restores LR emergence, indicating that elevated SA levels may be responsible for the mutant's developmental defects. MutMap analysis and allelism tests confirmed that the phenotypes of the dlr1-1 mutant results from the loss of the Na+/H+ antiporter, ZmNHX7. Additionally, the application of NaCl exacerbates the dlr1-1 mutant phenotype, suggesting that the root defects in dlr1-1 mutant depend on ion homoeostasis. In conclusion, our findings demonstrate that maize DLR1/NHX7 is essential for root development under potassium deprivation.

Strain variation in Candida albicans glycolytic gene regulation.

Central carbon metabolism is vital for the proliferation of Candida albicans, a fungus that is prominent as a commensal and pathogen. Glycolytic genes are activated by overlapping activities of the transcription factors Tye7 and Gal4, as shown by studies in the SC5314 genetic background. However, regulatory relationships can vary among C. albicans isolates. Here, we analyzed Tye7- and Gal4-related phenotypes in five diverse clinical isolates of C. albicans. We tested growth properties and gene expression impact through Nanostring profiling and, for the two strains SC5314 and P87, RNA sequencing. Our results lead to three main conclusions. First, the functional redundancy of Tye7 and Gal4 for glycolytic gene activation is preserved among all strains tested. Second, at the gene expression level, strain P87 is an outlier with regard to tye7Δ/Δ impact, and strain SC5314 is an outlier with regard to gal4Δ/Δ impact. Third, while Gal4 is well known to be dispensable for induction of the GAL1, GAL7, and GAL10 galactose-specific metabolic genes, we find that gal4Δ/Δ mutants of several strains have a mild galactose fermentation defect, as assayed by growth on galactose with the respiration inhibitor antimycin A. Our findings indicate that even a central metabolic regulatory network is subject to strain variation and illustrates an unexpected genotype-phenotype relationship.The fungal commensal and pathogen Candida albicans rely upon metabolic flexibility to colonize and infect host niches. Central carbon metabolism is governed by two regulators, Tye7 and Gal4, as defined in the reference strain SC5314. Here, we have explored the impact of Tye7 and Gal4 on carbon utilization and gene expression across five diverse C. albicans clinical isolates. Novel aspects of this study are the finding that even a central metabolic regulatory network is subject to strain variation and the observation of an unexpected mutant phenotype.

Functions of the Sinorhizobium meliloti LsrB Substrate-Binding Domain in Oxidized Glutathione Resistance, Alfalfa Nodulation Symbiosis, and Growth.

To successfully colonize legume root nodules, rhizobia must effectively evade host-generated reactive oxygen species (ROS). LsrB, a redox regulator from Sinorhizobium meliloti, is essential for symbiosis with alfalfa (Medicago sativa). The three cysteine residues in LsrB's substrate domain play distinct roles in activating downstream redox genes. The study found that LsrB's substrate-binding domain, dependent on the cysteine residue Cys146, is involved in oxidized glutathione (GSSG) resistance and alfalfa nodulation symbiosis. LsrB homologues from other rhizobia, with Cys172/Cys238 or Cys146, enhance GSSG resistance and complement lsrB mutant's symbiotic nodulation. Substituting amino acids in Azorhizobium caulinodans LsrB with Cys restores lsrB mutant phenotypes. The lsrB deletion mutant shows increased sensitivity to NCR247, suggesting an interaction with host plant-derived NCRs in alfalfa nodules. Our findings reveal that the key cysteine residue in the LsrB's substrate domain is vital for rhizobium-legume symbiosis.

Genome-wide analysis of glutathione S-transferase genes in four Prunus species and the function of PmGSTF2, activated by PmMYBa1, in regulating anthocyanin accumulation in Prunus mume

Glutathione S-transferases (GSTs) are proteases with multiple physiological functions and play an important role in plant responses to abiotic stresses. Nevertheless, there is a paucity of systematic research on GST genes in Prunus genus. Here, 330 GST genes in four Prunus species were identified for the first time and classified into eight subgroups based on protein sequence and conserved structure, among which Tau subfamily genes had the largest number. The amino acid lengths of GST-encoded proteins in the four species ranged from 66 to 1152 aa, most of which were soluble proteins and located in the cytoplasm and chloroplasts. The GST family was propelled by tandem duplications, yet robust purifying selection constrained its divergence. Conserved motif and domain analysis revealed that the majority of PmGSTs exhibited a highly conserved GST-N structure. The expression pattern of PmGSTs exhibited tissue specificity and spatiotemporal specificity. qRT-PCR validated the transcriptome results and 11 genes were differentially expressed in varieties with different flower and stem colors. In addition, we discovered an anthocyanin-related gene PmGSTF2, which can effectively restore the anthocyanin and proanthocyanidin deficiency-related phenotypes of the Arabidopsis tt19 mutant. Recombinant PmGSTF2 enhanced the water solubility of cyanidin and cyanidin-3-O-glucoside in vitro. Moreover, PmMYBa1 could directly bind to the promoter of PmGSTF2 and activate its expression. The findings revealed that GSTs were preserved in Prunus species and that PmGSTF2 was critical in regulating anthocyanin accumulation.

CLAVATA3 INSENSITIVE RECEPTOR KINASEs regulate lateral root initiation and spacing in Arabidopsis.

The root system architecture is very critical for plants to adapt to ever-changing environmental stimulations and is largely affected by lateral roots (LRs). Therefore, how plants regulate LR initiation and spacing is a key point for root system development. Previous studies have shown that RECEPTOR-LIKE KINASE 7 (RLK7) and its ligand TARGET OF LBD SIXTEEN 2 (TOLS2) control the initiation and spacing of LRs. However, the molecular mechanism underlying the perception and transduction of the TOLS2 signal by RLK7 remains to be elucidated. In this study, we explored whether CLAVATA3 INSENSITIVE RECEPTOR KINASEs (CIKs) are critical signaling components during Arabidopsis (Arabidopsis thaliana) LR development by investigating phenotypes of cik mutants and examining interactions between CIKs and members of the RLK7-mediated signaling pathway. Our results showed that high-order cik mutants generated more LRs because of more LR initiation and defective LR spacing. The cik mutants showed reduced sensitivity to applied TOLS2 peptides. TOLS2 application enhanced the interactions between CIKs and RLK7 and the RLK7-dependent phosphorylation of CIKs. In addition, overexpression of transcription factor PUCHI and constitutive activation of MITOGEN-ACTIVATED PROTEIN KINASE KINASE 4 (MKK4) and MKK5 partially rescued the spacing defects of LRs in cik and rlk7-3 mutants. Moreover, we discovered that auxin maximum in pericycle cells altered subcellular localization of CIKs to determine lateral root founder cells. These findings revealed that CIKs and RLK7 function together to perceive the TOLS2 signal and regulate LR initiation and spacing through the MKK4/5-MPK3/6-PUCHI cascade.

The functional roles of zebrafish HoxA- and HoxD-related clusters in the pectoral fin development

The paralogs 9–13 Hox genes in mouse HoxA and HoxD clusters are critical for limb development. When both HoxA and HoxD clusters are deleted in mice, significant limb truncation is observed compared to the phenotypes of single and compound mutants of Hox9-13 genes in these clusters. In zebrafish, mutations in hox13 genes in HoxA- and HoxD-related clusters result in abnormal morphology of pectoral fins, homologous to forelimbs. However, the effect of the simultaneous deletions of entire HoxA- and HoxD-related clusters on pectoral fin development remains unknown. Here, we generated mutants with several combinations of hoxaa, hoxab, and hoxda cluster deletions and analyzed the pectoral fin development. In hoxaa−/−;hoxab−/−;hoxda−/− larvae, the endoskeletal disc and the fin-fold are significantly shortened in developing pectoral fins. In addition, we show that this anomaly is due to defects in the pectoral fin growth after the fin bud formation. Furthermore, in the surviving adult mutants, micro-CT scanning reveals defects in the posterior portion of the pectoral fin which is thought to represent latent regions of the limb. Our results further support that the functional role of HoxA and HoxD clusters is conserved in the paired appendage formation in bony fishes.

C. elegans epicuticlins define specific compartments in the apical extracellular matrix and function in wound repair.

The apical extracellular matrix (aECM) of external epithelia often contains lipid-rich outer layers that contribute to permeability barrier function. The external aECM of nematodes is known as the cuticle and contains an external lipid-rich layer, the epicuticle. Epicuticlins are a family of tandem repeat cuticle proteins of unknown function. Here, we analyze the localization and function of the three C. elegans epicuticlins (EPIC proteins). EPIC-1 and EPIC-2 localize to the surface of the cuticle near the outer lipid layer, as well as to interfacial cuticles and adult-specific struts. EPIC-3 is expressed in dauer larvae and localizes to interfacial aECM in the buccal cavity. Skin wounding in the adult induces epic-3 expression, and EPIC proteins localize to wound sites. Null mutants lacking EPIC proteins are viable with reduced permeability barrier function and normal epicuticle lipid mobility. Loss of function in epic genes modifies the skin blistering phenotypes of bli mutants and reduces survival after skin wounding. Our results suggest EPIC proteins define specific cortical compartments of the aECM and promote wound repair.

Full-Length Transcriptome Analysis and Characterization of DFRs Involved in the Formation of Anthocyanin in Allium wallichii

Allium wallichii is famous for its reddish-purple flowers, which can be utilized as cut flowers and garden landscaping. Flower color is mainly determined by flavonoids, betalains, carotenoids, as well as other pigments. However, there is no research on the color formation mechanism in A. wallichii, which restricts its genetic improvement and development of superior varieties. The flower of A. wallichii was collected for full-length transcriptome sequencing and metabolome analysis using PacBio SMART and UPLC-MS, respectively. A total of 45 anthocyanins were detected in its flower, and 75,778 transcripts of 107,208 non-redundant transcripts were annotated. Then, two AwDFRs were cloned and characterized using bioinformatics tools. Enzyme activity assays revealed that both AwDFR1 and AwDFR2 possessed DFR activity in vitro that only accepted DHQ and DHM as substrates, except for DHK. Finally, physiological results showed that AwDFR1 and AwDFR2 could restore the lacking phenotypes of Arabidopsis tt3 mutant and increase the content of anthoycanin in tobacco petals. The anthocyanins and transcriptome in A. wallichii were firstly reported, and AwDFR1 and AwDFR2 are key enzymes participating in the biosynthesis of anthocyanins. This research provides important guidance for future key gene mining, color improvement, and horticultural breeding in A. wallichii.

Assessing the Role of AtGRP7 Arginine 141, a Target of Dimethylation by PRMT5, in Flowering Time Control and Stress Response.

PROTEIN ARGININE METHYLTRANSFERASES (PRMTs) catalyze arginine (R) methylation that is critical for transcriptional and post-transcriptional gene regulation. In Arabidopsis, PRMT5 that catalyzes symmetric R dimethylation is best characterized. PRMT5 mutants are late-flowering and show altered responses to environmental stress. Among PRMT5 targets are Arabidopsis thaliana GLYCINE RICH RNA BINDING PROTEIN 7 (AtGRP7) and AtGRP8 that promote the transition to flowering. AtGRP7 R141 has been shown to be modified by PRMT5. Here, we tested whether this symmetric dimethylation of R141 is important for AtGRP7's physiological role in flowering time control. We constructed AtGRP7 mutant variants with non-methylable R141 (R141A, R141K). Genomic clones containing these variants complemented the late-flowering phenotype of the grp7-1 mutant to the same extent as wild-type AtGRP7. Furthermore, overexpression of AtGRP7 R141A or R141K promoted flowering similar to overexpression of the wild-type protein. Thus, flowering time does not depend on R141 and its modification. However, germination experiments showed that R141 contributes to the activity of AtGRP7 in response to abiotic stress reactions mediated by abscisic acid during early development. Immunoprecipitation of AtGRP7-GFP in the prmt5 background revealed that antibodies against dimethylated arginine still recognized AtGRP7, suggesting that additional methyltransferases may be responsible for modification of AtGRP7.

Foxe1 mutant zebrafish show indications of a hypothyroid phenotype and increased sensitivity to ethanol for craniofacial malformations.

FOXE1 mutations in humans are associated with cleft palate and hypothyroidism. We previously developed a foxe1 mutant zebrafish demonstrating mineralization defects in larvae. In the present study, we investigate the thyroid status and skeletal phenotype of adult foxe1 mutants. Mutant fish have increased expression of tshβ in the pituitary, and of hepatic dio1 and dio2. In plasma, we found higher Mg levels. Together these findings are indicative of hypothyroidism. We further observed mineralization defects in scales due to enhanced osteoclast activity as measured by increased expression levels of tracp, ctsk, and rankl. Gene-environment interactions in the etiology of FOXE1-related craniofacial abnormalities remain elusive, which prompts the need for models to investigate genotype-phenotype associations. We here investigated whether ethanol exposure increases the risk of developing craniofacial malformations in foxe1 mutant larvae that we compared to wild types. We found in ethanol-exposed mutants an increased incidence of developmental malformations and marked changes in gene expression patterns of cartilage markers (sox9a), apoptotic markers (casp3b), retinoic acid metabolism (cyp26c1), and tissue hypoxia markers (hifaa, hifab). Taken together, this study shows that the foxe1 mutant zebrafish recapitulates phenotypes associated with FOXE1 mutations in human patients and a clear foxe1-ethanol interaction.

CRISPR/Cas9-mediated knockout of the abdominal-B homeotic gene in the global pest, fall armyworm (Spodoptera frugiperda).

The Homeotic complex (Hox) genes play a crucial role in determining segment identity and appendage morphology in bilaterian animals along the antero-posterior axis. Recent studies have expanded to agricultural pests such as fall armyworm (FAW), scientifically known as Spodoptera frugiperda J. E. Smith (Lepidoptera: Noctuidae), which significantly threatens global agricultural productivity. However, the specific role of the hox gene Sfabd-B in FAW remains unexplored. This research investigates the spatial and temporal expression patterns of Sfabd-B in various tissues at different developmental stages using quantitative real-time polymerase chain reaction (qRT-PCR). Additionally, we explored the potential function of the Sfabd-B gene located in the FAW genome using CRISPR/Cas9 technology. The larval mutant phenotypes can be classified into three subgroups as compared with wild-type individuals, that is, an excess of pedis in the posterior abdomen, deficient pedis due to segmental fusion and deviations in the posterior abdominal segments. Importantly, significant differences in mutant phenotypes between male and female individuals were also evident during the pupal and adult phases. Notably, both the decapentaplegic (dpp) and cuticular protein 12 (cp 12) genes displayed a substantial marked decrease in expression levels in the copulatory organ of male mutants and the ovipositor of female mutants compared with the wild type. These findings highlight the importance of Sfabd-B in genital tract patterning, providing a potential target for improving genetic control.

An osmesl mutant delayed rice leaf senescence through inhibiting cell death by OsBI-1

Leaf senescence following heading in rice is subject to rigorous regulation, with many of the underlying control mechanisms remaining largely unknown. In this study, we identified a novel gene, OsMESL, which exerts a positive regulatory effect on leaf senescence in rice. The T-DNA insertion mutant known as osmesl and RNA interference plants displayed a phenotype characterized by stay-green after heading. Genetic analysis indicated that the mutant phenotype could be rescued through complementation, while the overexpression of OsMESL accelerated leaf senescence after heading, underscoring OsMESL's positive regulatory role in rice leaf senescence. Subsequent investigations revealed that OsMESL modulates the process of cell death by influencing the stability of its interacting protein, the cell death suppressor OsBI-1, thereby governing leaf senescence. Furthermore, the leaves of the osmesl mutant exhibited a delayed reduction in photosynthesis, along with increased grain length and 1000-grain weight. In conclusion, we identified OsMESL as a novel positive regulator of leaf senescence in rice, which likely participates in leaf senescence through the mediation of cell death by OsBI-1, resulting in the phenotype of stay-green in the osmesl mutant after heading.

Plant BCL-Domain Homologues play a conserved role in SWI/SNF complex stability.

The SWItch/Sucrose Non-Fermenting (SWI/SNF) complexes are evolutionarily conserved, ATP-dependent chromatin remodelers crucial for multiple nuclear functions in eukaryotes. Recently, plant BCL-Domain Homolog (BDH) proteins were identified as shared subunits of all plant SWI/SNF complexes, significantly impacting chromatin accessibility and various developmental processes in Arabidopsis. In this study, we performed a comprehensive characterization of bdh mutants, revealing a previously overlooked impact on hypocotyl cell elongation. Through detailed analysis of BDH domains, we identified a plant-specific N-terminal domain that facilitates the interaction between BDH and the rest of the complex. Additionally, we uncovered the critical role of the BDH β-hairpin domain, which is phylogenetically related to metazoan BCL7 SWI/SNF subunits. While phylogenetic analyses did not identify BDH/BCL7 orthologs in fungi, structure prediction modeling demonstrated strong similarities between the SWI/SNF catalytic modules of plants, animals, and fungi, and revealed the yeast Rtt102 protein as a structural homolog of BDH and BCL7. This finding is supported by the ability of Rtt102 to interact with the Arabidopsis catalytic module subunit ARP7 and partially rescue the bdh mutant phenotypes. Further experiments revealed that BDH promotes the stability of the ARP4-ARP7 heterodimer, leading to the partial destabilization of ARP4 in the SWI/SNF complexes. In summary, our study unveils the molecular function of BDH proteins in plant SWI/SNF complexes and suggests that β-hairpin-containing proteins are evolutionarily conserved subunits crucial for ARP heterodimer stability and SWI/SNF activity across eukaryotes.

Dual role of the S5 segment in type 1 ryanodine receptor channel gating

The type 1 ryanodine receptor (RyR1) is a Ca2+ release channel in the sarcoplasmic reticulum that is essential for skeletal muscle contraction. RyR1 forms a channel with six transmembrane segments, in which S5 is the fifth segment and is thought to contribute to pore formation. However, its role in channel gating remains unclear. Here, we performed a functional analysis of several disease-associated mutations in S5 and interpreted the results with respect to the published RyR1 structures to identify potential interactions associated with the mutant phenotypes. We demonstrate that S5 plays a dual role in channel gating: the cytoplasmic side interacts with S6 to reduce the channel activity, whereas the luminal side forms a rigid structural base necessary for S6 displacement in channel opening. These results deepen our understanding of the molecular mechanisms of RyR1 channel gating and provide insight into the divergent disease phenotypes caused by mutations in S5.

OsFAD1-OsMYBR22 modulates clustered spikelet through regulating BRD3 in rice.

The phenotype of rice clustered spikelet mutants results from the upregulation of the FAD/NAD(P)-binding oxidoreductase family gene OsFAD1. Enhanced interaction between OsFAD1 and the transcription factor OsMYBR22 leads to the upregulation of the spikelet clustering-related BR catabolic gene BRD3.

Analysis of randomly mutated AlSRKb genes reveals that most loss-of-function mutations cause defects in plasma membrane localization.

Only very limited information is available on why some nonsynonymous variants severely alter gene function while others have no effect. To identify the characteristic features of mutations that strongly influence gene function, this study focused on SRK which encodes a highly polymorphic receptor kinase expressed in stigma papillary cells that underlies a female determinant of self-incompatibility in Brassicaceae. A set of 300 Arabidopsis thaliana transformants expressing mutated SRKb from A. lyrata was constructed using error-prone PCR and the genotype and self-incompatibility phenotype of each transformant were determined. Almost all the transformants showing the self-incompatibility defect contained mutations in AlSRKb that altered localization to the plasma membrane. The observed mutations occurred in amino acid residues that were highly conserved across S haplotypes and whose predicted locations were in the interior of the protein. Our findings suggested that mutations causing the self-incompatibility defect were more likely to result from changes to AlSRKb biosynthesis than from loss of AlSRKb function. In addition, we examined whether the RandomForest and Extreme Gradient Boosting methods could predict the self-incompatibility phenotypes of SRK mutants.

Anti-Müllerian hormone deficiency leads to two distinct ovarian phenotypes with different alterations of sex hormones in gynogenetic carp

Anti-Müllerian hormone (Amh) plays conserved roles in gonadal development and function in vertebrates. Previous studies have demonstrated that amh deficiency results in severe gonadal hypertrophy in teleost. Here, we report a novel phenotype of gonadal atrophy caused by amh knockout in fish. Firstly, it was found that the amh gene had three alleles and was expressed in the cytoplasm of follicle cells in gynogenetic amphiploid gibel carp (Carassius gibelio). Subsequently, we constructed a mutant line of amh and identified two distinct ovarian abnormalities in gibel carp. Most mutants exhibited hypertrophic ovaries with increased proliferation and decreased differentiation of early germ cells, which is consistent with previously reported findings. Intriguingly, a small number of mutants displayed atrophic ovaries with decreased proliferation and premature differentiation of germ cells, which represents a novel phenotype in fish species. Transcriptome analysis revealed that these two distinct ovaries of amh mutants showed different alterations in the sex hormone biosynthesis pathway, when compared with wild-type ovaries. Furthermore, diverse gonadal sex hormone levels were also detected in these two abnormal ovaries, which were consistent with the transcriptional expressions of corresponding genes in the sex hormone synthesis pathway. These findings identify a novel atrophic ovarian phenotype of amh mutants and confirm the amh function in controlling the balance between proliferation and differentiation of germ cells in fish.

Unveiling the genetic basis and metabolic rewiring behind the galactose-positive phenotype in a Streptococcus thermophilus mutant

Streptococcus thermophilus (S. thermophilus) is a widely used starter culture in dairy fermentation, but most strains are galactose-negative and only metabolize glucose from lactose hydrolysis. In this study, we aimed to uncover the mechanisms underlying the acquisition of a stable galactose-positive (Gal+) phenotype in a mutant strain of S. thermophilus IMAU10636. By treating the wild-type strain with the mutagenic agent N-methyl-N-nitro-N-nitrosoguanidine, we successfully isolated a Gal+ mutant, S. thermophilus IMAU10636Y. Comparative enzyme activity assays revealed that the mutant exhibited higher β-galactosidase and galactokinase activities, but lower glucokinase and pyruvate kinase activities compared to the wild-type. High-performance liquid chromatography analysis confirmed the mutant’s enhanced ability to utilize lactose and galactose, leading to increased glucose secretion. Integrated genome and transcriptomics analyses provided deeper insights into the underlying genetic and metabolic mechanisms. We found that the metabolism regulatory network of the glycolysis / Leloir pathway was altered in the mutant, possibly due to the upregulation of the gene expression in the galR-galK intergenic region. This likely led to increased RNA polymerase binding and transcription of the gal operon, ultimately promoting the Gal+ phenotype. Additionally, we identified a mutation in the scrR gene, encoding a LacI family transcriptional repressor, which also contributed to the Gal+ phenotype. These findings offer new perspectives on the metabolic rewiring and regulatory mechanisms that enable S. thermophilus to acquire the ability to metabolize galactose. This knowledge can inform strategies for engineering and selecting Gal+ strains with desirable fermentation characteristics for dairy applications.

MYB Transcription Factor CDC5 Activates CBF3 Expression to Positively Regulates Freezing Tolerance via Cooperating With ICE1 and Histone Modification in Arabidopsis.

The freezing temperature greatly limits the growth, development and productivity of plants. The C-repeat/DRE binding factor (CBF) plays a major role in cold acclimation, enabling plants to increase their freezing tolerance. Notably, the INDUCER OF CBF EXPRESSION1 (ICE1) protein has garnered attention for its pivotal role in bolstering plants' resilience against freezing through transcriptional upregulation of DREB1A/CBF3. However, the research on the interaction between ICE1 and other transcription factors and its function in regulating cold stress tolerance is largely inadequate. In this study, we found that a R2R3 MYB transcription factor CDC5 interacts with ICE1 and regulates the expression of CBF3 by recruiting RNA polymerase II, overexpression of ICE1 can complements the freezing deficient phenotype of cdc5 mutant. CDC5 increases the expression of CBF3 in response to freezing. Furthermore, CDC5 influences the expression of CBF3 by altering the chromatin status through H3K4me3 and H3K27me3 modifications. Our work identified a novel component that regulates CBF3 transcription in both ICE1-dependent and ICE1-independent manner, improving the understanding of the freezing signal transduction in plants.

Identification of Transposon Insertion Sites in MaizeMu-Tagged Mutants Using Mu-Seq

Mutator(Mu) transposons facilitate untargeted insertional mutagenesis in maize by moving within the genome and disrupting genes. Such an approach has been used to generate collections such as theBonnMuresource, aMu-tagged maize population for functional genomics studies. Mutant-Seq (Mu-Seq) is a sequencing-based method for the high-throughput identification and mapping ofMuinsertion sites. The approach involves the construction of multiplexed sequencing libraries (known as Mu-Seq libraries) fromMu-tagged populations, followed by high-throughput sequencing and data processing using the Mu-Seq Workflow Utility (MuWU) tool, to determine the location ofMuinsertions. Here, we provide a detailed protocol for Mu-Seq, from the generation of the maizeMu-tagged mutant population to data analysis. Researchers can use this approach to develop mutant collections customized to specific genetic backgrounds of interest, which can aid in characterizing genotype-specific mutations and identifying candidate genes linked to visible mutant phenotypes.