Mutant Lines Research Articles

OsAlR3 regulates aluminum tolerance through promoting the secretion of organic acids and the expression of antioxidant genes in rice

In acidic soils, aluminum (Al) toxicity inhibits the growth and development of plant roots and affects nutrient and water absorption, leading to reduced yield and quality. Therefore, it is crucial to investigate and identify candidate genes for Al tolerance and elucidate their physiological and molecular mechanisms under Al stress. In this study, we identified a new gene OsAlR3 regulating Al tolerance, and analyzed its mechanism from physiological, transcriptional and metabolic levels. Compared with the WT, malondialdehyde (MDA) and hydrogen peroxide (H2O2) content were significantly increased, superoxide dismutase (SOD) activity and citric acid (CA) content were significantly decreased in the osalr3 mutant lines when exposed to Al stress. Under Al stress, the osalr3 exhibited decreased expression of antioxidant-related genes and lower organic acid content compared with WT. Integrated transcriptome and metabolome analysis showed the phenylpropanoid biosynthetic pathway plays an important role in OsAlR3-mediated Al tolerance. Exogenous CA and oxalic acid (OA) could increase total root length and enhance the antioxidant capacity in the mutant lines under Al stress. Conclusively, we found a new gene OsAlR3 that positively regulates Al tolerance by promoting the chelation of Al ions through the secretion of organic acids, and increasing the expression of antioxidant genes.

Assessment of grain quality traits in mutant lines of winter barley

This study aimed to evaluate the variation among mutant lines derived from the winter barley variety Gigga, focusing on crucial feed quality traits such as protein, lysine, ash, fat, fiber content, and 1000-grain weight. Significant differences were identified in specific traits among the mutant lines, revealing promising candidates for further breeding programs. Mutant lines Gigga-25, Gigga-26, and Gigga-29 exhibited significantly higher protein content in their grains compared to the parent variety Gigga. In addition, Gigga-10 demonstrated elevated lysine content, while Gigga-4, Gigga-12, and Gigga-20 displayed increased 1000-grain weight. Of particular interest was Gigga-20, standing out for its exceptional combination of high protein content and favorable 1000-grain weight. This dual advantage positions Gigga-20 as a prime candidate for further investigation and potential integration into breeding programs, aiming to develop barley varieties with superior feed quality. Furthermore, a promising balance was observed in mutant lines Gigga-25 and Gigga-28, showcasing favorable proportions of protein and lysine content. These mutants present additional options for incorporation into breeding initiatives directed at enhancing the overall nutritional quality of feed grains.

Breeding for Resistance to Biotic Stresses in Crops: Conventional to Genome Editing

Biotic stress is caused by fungi, bacteria, viruses, insects, nematodes as well weeds and it significantly reduces crop yield globally. For instance, the outbreak of a new strain of stripe rust in wheat worldwide, the emergence of white scale insect affecting mango in Ethiopia, the newly emerged Maize Lethal Necrosis viral disease in maize, and invasive fall armyworm insect are devastating maize crop and causing yield loss in Africa. To reduce the yield losses due to biotic stresses, the development of resistant variety and integrated insect pest approach is the way forward for managing disease and insects at respective agroecologies. Thus, this review paper discussed on conventional breeding methods and molecular-assisted selection for breeding resistance to foliar disease in major cereal crops. Wide array of germplasm such as landraces, recombinant inbred lines, pure lines, Double haploid lines, elite lines, multi-parent population, mutant lines, introgressed lines, hybrids, open population variety and wild relative can be used as source germplasm and should be screened under artificial inoculation and or at hotspot areas to develop disease resistance variety. Many maize inbred lines and hybrids showed resistance to turcicum leaf blight, grey leaf spot and common rust diseases and indicating that these genotypes have carrying genes/favorable alleles for multiple disease resistance and it is possible to develop variety resistance to fungi foliar disease in maize. Similarly, several advanced lines and some varieties showed resistance to strip and leaf rust in wheat. However, host plant resistance could be broken down due to new emerging race pathogens. Thus, conventional breeding and molecular screening should be integrated for resistant variety development. Indeed, Marker-assisted selection through backcrossing, gene pyramiding, combined Genome-Wide Association, and transcriptome approach is useful to identify candidate genes and resistant parents in crops. .......

Lectin Receptor-Like Protein Kinase OsNRFG6 is Required for Embryo Sac Development and Fertilization in Neo-Tetraploid Rice

Great yield-enhancing prospects of autotetraploid rice was restricted by various polyploidy-induced reproductive dysfunction. To surmount these challenges, our group has generated a series of valuable fertile tetraploid lines (denoted as neo-tetraploid rice) through 20-year efforts. With this context, a G-type lectin receptor-like kinase, OsNRFG6, was identified as a pivotal factor associated with reproductive regulation in neo-tetraploid rice. Nevertheless, it is still elusive about a comprehensive understanding of its precise functional roles and underlying molecular mechanisms during reproduction of neo-tetraploid rice. Here, we demonstrated that OsNRFG6 executed a constitutive expression pattern and encoded proteins localizing in perinucleus and endoplasmic reticulum. Subsequently, four independent mutant lines of OsNRFG6 within neo-tetraploid rice background were further identified, all displaying low seed-setting rate due to abortive embryo sacs and defective double fertilization. RNA-seq and RT-qPCR revealed a significant down-regulation of OsNRFG6 and female reproductive genes such as OsMEL1 and LOG in ovaries prior to and post-fertilization, attributing this effect to OsNRFG6 mutation. Furthermore, through yeast-two hybrids, bimolecular fluorescence complementation assays, and luciferase complementation imaging assays, it was determined that OsNRFG6 could interact with itself and two female reproductive proteins (LOG and OsDES1) to form protein complexes. These results elucidate the reproductive functions and molecular pathway governed by OsNRFG6 in regulating fertility of neo-tetraploid rice, offering insights into molecular understanding of fertility improvement in polyploid rice.

Identification of Drought Tolerant Fenugreek Mutants at Seedling Stage Through Multi-Trait Genotype-Ideotype Distance Index (MGIDI)

The top-performing mutants from the M2 generation, induced by a combination of γ radiation and EMS treatment, underwent screening for drought tolerance at the seedling stage in the M3 generation under controlled laboratory conditions. This investigation aimed to assess their potential for drought tolerance. The top 15 high-yielding mutants were assessed under both normal (control) and induced drought conditions (10% PEG solution). Utilizing the MGIDI method, the evaluation provided valuable insights into selection traits and identified mutants with potential drought tolerance. Under normal conditions, mutants M3-181 and M3-149 exhibited superior performance in seedling growth traits, with M3-149 being the top performer under induced drought conditions, followed by M3-181. Their proximity to the red-cutline suggests promising potential that warrants further investigation. Furthermore, these findings indicate that mutant lines displayed superior multi-trait performance compared to the parent cultivar. Specifically, mutants M3-181, M3-149, and M3-29 ranked higher than the parent cultivar under normal conditions, while mutants M3-149, M3-181, and M3-168 surpassed the parent cultivar RMt-1 under induced drought conditions. This underscores the effectiveness of mutation breeding, particularly through the induction of mutations using γ radiation and EMS, as an efficient strategy not only for generating genetic diversity but also for producing variants superior to the parent cultivar. To the best of our knowledge, this is first report made on mutant selection in fenugreek for drought tolerance at seedling stage, using the MGIDI approach.

Isolation and evaluation of novel male sterile and self‐incompatible mutant lines of mungbean (Vigna radiata L.)

AbstractHeterosis has long been harnessed in crop production, and while crop fertility lays the groundwork for leveraging heterosis, there remains a dearth of comprehensive data regarding genic male sterility in mungbean. Mungbean (Vigna radiata (L.) Wilczek), being a self‐pollinating crop, exhibits significant hybrid vigour in F1 hybrid seed yield, suggesting the potential for developing hybrid varieties to propel mungbean yield beyond existing plateaus. A new male sterile mutant was induced, isolated, stabilized and evaluated, via gamma irradiation at a rate of 600 Gy, identified in the M3 generation of the GM 4 accession. A uniform male sterile line was crossbred with the wild‐type parental plant to explore the inheritance pattern of male sterility. Pollen sterility was validated through the acetocarmine test, while stigma viability was ascertained using the hydrogen peroxide. While all F1 plants displayed fertile flowers, the F2 generation showed a clear 3:1 segregation ratio for fertile to male sterile plants, indicating the control of male sterility by a single recessive gene, mms. Also, a novel existence of a truncated stigma nestled within the anther column inhibits the effective reception of pollen during anthesis, offering potential in reducing emasculation time by a slight cut on the flower bud and enhancing cross pollination during hybrid seed production. The isolation of these two mutants is poised to significantly advance the global mungbean hybrid breeding programme.

Generation of New β-Conglycinin-Deficient Soybean Lines by Editing the lincRNA lincCG1 Using the CRISPR/Cas9 System.

Soybean β-conglycinin is a major allergen that adversely affects the nutritional properties of soybean. Soybean deficient in β-conglycinin is associated with low allergenicity and high nutritional value. Long intergenic noncoding RNAs (lincRNAs) regulate gene expression and are considered important regulators of essential biological processes. Despite increasing knowledge of the functions of lincRNAs, relatively little is known about the effects of lincRNAs on the accumulation of soybean β-conglycinin. The current study presents the identification of a lincRNA lincCG1 that was mapped to the intergenic noncoding region of the β-conglycinin α-subunit locus. The full-length lincCG1 sequence was cloned and found to regulate the expression of soybean seed storage protein (SSP) genes via both cis- and trans-acting regulatory mechanisms. Loss-of-function lincCG1 mutations generated using the clustered regularly interspaced short palindromic repeats/CRISPR-associated protein 9 (CRISPR/Cas9) system led to the deficiency of the allergenic α'-, α-, and β-subunits of soybean β-conglycinin as well as higher content of proteins, sulfur-containing amino acids, and free arginine. The dominant null allele LincCG1, and consequently, the β-conglycinin-deficient phenotype associated with the lincCG1-gene-edited line was stably inherited by the progenies in a Mendelian fashion. The dominant null allele LincCG1 may therefore be exploited for engineering/developing novel hypoallergenic soybean varieties. Furthermore, Cas9-free and β-conglycinin-deficient homozygous mutant lines were obtained in the T1 generation. This study is the first to employ the CRISPR/Cas9 technology for editing a lincRNA gene associated with the soybean allergenic protein β-conglycinin. Moreover, this study reveals that lincCG1 plays a crucial role in regulating the expression of the β-conglycinin subunit gene cluster, besides highlighting the efficiency of employing the CRISPR/Cas9 system for modulating lincRNAs, and thereby regulating soybean seed components.

Phenanthrene-Induced Cytochrome P450 Genes and Phenanthrene Tolerance Associated with Arabidopsis thaliana CYP75B1 Gene.

Polycyclic aromatic hydrocarbons (PAHs) form an important group of organic pollutants due to their distribution in the environment and their carcinogenic and/or mutagenic effects. In order to identify at the molecular level some of the players in the biodegradation and tolerance response to PAHs in plants, we have phenotyped 32 Arabidopsis thaliana T-DNA mutant lines corresponding to 16 cytochrome P450 (CYP) genes that showed to be differentially expressed under contrasted stress conditions induced by phenanthrene, a 3-ring PAH. This screening has allowed us to identify CYP75B1 (At5g07990) T-DNA mutants as the only ones being sensitive to phenanthrene-induced stress, supporting that CYP75B1 protein is necessary for PAH tolerance. CYP75B1 codes for a 3'flavonol hydroxylase. CYP75B1 gene was heterologously expressed on yeast in order to investigate whether it affects the A. thaliana response to phenanthrene by participating in its metabolization. Heterologously-produced CYP75B1 enzyme shows to be catalytically efficient against its physiological substrates (e.g., naringenin) but unable to metabolize phenanthrene or 9-phenanthrenol. In contrast, CYP75B1 seems rather involved in phenanthrene tolerance as a crucial element by regulating concentration of antioxidants through the production of 3'-hydroxylated flavonoids such as quercetin and cyanidin. In particular, we report a highly increased generation of reactive oxygen species (H2O2 and singlet oxygen) in cyp75b1 mutants compared to control plants in response to phenanthrene treatment. Overall, CYP75B1 shows to play an important role in the response to the deleterious effects of phenanthrene exposure and this is related to oxidative stress sensitivity rather than metabolization.

Application of SNP Markers to Identify Genetic Variation on Mutant Lines of Adan-Krayan, Special Local Rice from North Kalimantan

Adan rice, a local rice from the Krayan highland of North Kalimantan is marked by its fine taste that consumers like. However, this rice can only be planted once a year due to its long lifespan. Efforts have been made to develop a short-lived version of Adan, to enable it to be planted twice a year. Adan Kelabit was initially exposed to gamma radiation, which produced several mutants. Field selection identified eight mutants with shorter lifespans than the Adan. In this study, these eight mutants were observed further. SNP markers were carried out to confirm the occurrence of mutations, while observations of agronomic characters and grain quality were carried out to determine the most promising lines. The molecular study showed mutations found in all chromosomes of the mutants and differentiated them from the original Adan, with the similarity level ranging from 0.746 to 0.843. However, this closeness is not correlated to the dosage of the gamma rays applied. The majority of the mutants have shorter plant height and higher yield potential. On average, the mutant is 11.5% shorter on heading date, 22.69% higher on tiller number, 27.14% higher on grain weight, and 26.64% higher on other parameters of yield potential. Apart from the shorter heading date, each mutant has its superiorities. The A25 and A28 mutant lines have much higher yield potential while maintaining the same level of amylose content as the Adan rice. These two mutants can be considered for further development or dissemination for farmer adoption.

Investigating the mechanisms of isochorismate synthase: An approach to improve salicylic acid synthesis and increase resistance to Fusarium head blight in wheat

Salicylic acid (SA), a vital endogenous hormone, plays a crucial role in plant growth and the response to abiotic and biotic stress. Isochorismate synthase (ICS) and phenylalanine ammonia lyase (PAL) are critical rate-limiting enzymes for SA synthesis. Fusarium head blight (FHB) seriously threatens the safety of wheat production, but increasing the content of SA can enhance FHB resistance. However, the pathway of SA synthesis and regulation in wheat remains unknown. In this study, three wheat ICS (TaICSA, TaICSB, and TaICSD) were identified, and their functions were validated in vitro for isomerizing chorismate to isochorismate. The mutation of one or two homoeoalleles of TaICSA, TaICSB, and TaICSD in the wheat variety ‘Cadenza’ reduced SA levels under ultraviolet treatment and Fusarium graminearum infection, further enhancing sensitivity to FHB. Overexpression of TaICSA can significantly enhance SA levels and resistance to FHB. To further study SA synthesis pathways in wheat and avoid interference with pathogenicity related genes, the leaves of wild-type Cadenza and different TaICS mutant lines were subjected to ultraviolet treatment for transcriptomic analysis. The results showed that 37 PALs might be involved in endogenous SA synthesis, and 82 WRKY and MYB family transcription factors may regulate the expression of ICS and PAL. These results were further confirmed by RT-PCR. In conclusion, this study expands our knowledge of SA biosynthesis and identifies TaICSA, as well as several additional candidate genes that encode transcription factors for regulating endogenous SA levels, as part of an efficient strategy for enhancing FHB resistance in wheat.

CRIMP: a CRISPR/Cas9 insertional mutagenesis protocol and toolkit

Site-directed insertion is a powerful approach for generating mutant alleles, but low efficiency and the need for customisation for each target has limited its application. To overcome this, we developed a highly efficient targeted insertional mutagenesis system, CRIMP, and an associated plasmid toolkit, CRIMPkit, that disrupts native gene expression by inducing complete transcriptional termination, generating null mutant alleles without inducing genetic compensation. The protocol results in a high frequency of integration events and can generate very early targeted insertions, during the first cell division, producing embryos with expression in one or both halves of the body plan. Fluorescent readout of integration events facilitates selection of successfully mutagenized fish and, subsequently, visual identification of heterozygous and mutant animals. Together, these advances greatly improve the efficacy of generating and studying mutant lines. The CRIMPkit contains 24 ready-to-use plasmid vectors to allow easy and complete mutagenesis of any gene in any reading frame without requiring custom sequences, modification, or subcloning.

The trade-off between grain weight and grain number in wheat is explained by the overlapping of the key phases determining these major yield components.

Enhancing grain yield is a primary goal in the cultivation of major staple crops, including wheat. Recent research has focused on identifying the physiological and molecular factors that influence grain weight, a critical determinant of crop yield. However, a bottleneck has arisen due to the trade-off between grain weight and grain number, whose underlying causes remain elusive. In a novel approach, a wheat expansin gene, TaExpA6, known for its expression in root tissues, was engineered to express in the grains of the spring wheat cultivar Fielder. This modification led to increases in both grain weight and yield without adversely affecting grain number. Conversely, a triple mutant line targeting the gene TaGW2, a known negative regulator of grain weight, resulted in increased grain weight but decreased grain number, potentially offsetting yield gains. This study aimed to evaluate the two aforementioned modified wheat genotypes (TaExpA6 and TaGW2) alongside their respective wild-type counterparts. Conducted in southern Chile, the study employed a Complete Randomized Block Design with four replications, under well-managed field conditions. The primary metrics assessed were grain yield, grain number, and average grain weight per spike, along with detailed measurements of grain weight and dimensions across the spike, ovary weight at pollination (Waddington's scale 10), and post-anthesis expression levels of TaExpA6 and TaGW2. Results indicated that both the TaExpA6 and the triple mutant lines achieved significantly higher average grain weights compared to their respective wild types. Notably, the TaExpA6 line did not exhibit a reduction in grain number, thereby enhancing grain yield per spike. By contrast, the triple mutant line showed a reduced grain number per spike, with no significant change in overall yield. TaExpA6 expression peaked at 10 days after anthesis (DAA), and its effect on grain weight over the WT became apparent after 15 DAA. In contrast, TaGW2 gene disruption in the triple mutant line increased ovary size at anthesis, leading to improved grain weight above the WT from the onset of grain filling. These findings suggest that the trade-off between grain weight and number could be attributed to the overlapping of the critical periods for the determination of these traits.

Abstract PR014: Combinatorial genetic screens to map synthetic lethal interactions and identify new cancer drug targets in KRAS mutant cancers

Abstract The success of precision oncology depends on our ability to translate accumulating genomic data into actionable treatment options in a personalized manner. The first step requires the identification of a specific genomic signature, then matching this signature with the most effective therapy. Unfortunately, more often than not, we do not have a specific cancer drug that is effective for the mutation found in a specific tumor. We desperately need to identify new targets so that we can develop new drugs to deliver on the promise of precision cancer medicine. In addition, we need to develop a rational way to combine drugs that does not depend on trial and error. Combinatorial genetic screening CRISPR-Cas9 now allows us to identify synthetic lethal interactions in which simultaneous perturbation of two genes leads to cell death. However, multiplex gene editing to reveal these synergies between genes is a major challenge. For model systems like yeast, high-throughput methods and technologies have made it possible to create genetic networks with around 23 million double mutants (6000 genes x 6000 genes) and has resulted in the most detailed genetic interaction network to date consisting of ∼900,000 genetic interactions. This landscape of genetic interaction network of yeast has taken the scientific research community 15 years. In human cells, there are ∼20,000 genes and 400 million combinations, making this very complex network impossible to screen and test all these 400M combinations. To systematically interrogate such genetic interactions, we designed a dual CRISPR-Cas9 perturbation library targeting the top 1000 genes upregulated in KRAS mutant cancers (lung, pancreas and colon cancers). We performed these combinatorial double knockout screens on 100K (100x1000) unique gene pairs and identified 27 pairs whose co-disruption results in a loss of cellular fitness. We next validated those top synthetic lethal pairs of genes by performing secondary screens using CRISPR-Cas12a on more KRAS mutant and KRAS WT cell lines. Overall, our findings will provide insight into potential combinational targets in KRAS mutant tumors and will highlight the synthetic lethality effects that occurs among the novel targets, and other proliferation, metastasis, immune and metabolism modules. Citation Format: Rand Arafeh, Laura Chang, Lydia Sawyer, Helen Wang, James McFarland, Joshua Dempster, Peter DeWeirdt, John Doench, William C. Hahn. Combinatorial genetic screens to map synthetic lethal interactions and identify new cancer drug targets in KRAS mutant cancers [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Expanding and Translating Cancer Synthetic Vulnerabilities; 2024 Jun 10-13; Montreal, Quebec, Canada. Philadelphia (PA): AACR; Mol Cancer Ther 2024;23(6 Suppl):Abstract nr PR014.

NSE5 subunit interacts with distant regions of the SMC arms in the Physcomitrium patens SMC5/6 complex.

Structural maintenance of chromosome (SMC) complexes play roles in cohesion, condensation, replication, transcription, and DNA repair. Their cores are composed of SMC proteins with a unique structure consisting of an ATPase head, long arm, and hinge. SMC complexes form long rod-like structures, which can change to ring-like and elbow-bent conformations upon binding ATP, DNA, and other regulatory factors. These SMC dynamic conformational changes are involved in their loading, translocation, and DNA loop extrusion. Here, we examined the binding and role of the PpNSE5 regulatory factor of Physcomitrium patens PpSMC5/6 complex. We found that the PpNSE5 C-terminal half (aa230-505) is required for binding to its PpNSE6 partner, while the N-terminal half (aa1-230) binds PpSMC subunits. Specifically, the first 71 amino acids of PpNSE5 were required for binding to PpSMC6. Interestingly, the PpNSE5 binding required the PpSMC6 head-proximal joint region and PpSMC5 hinge-proximal arm, suggesting a long distance between binding sites on PpSMC5 and PpSMC6 arms. Therefore, we hypothesize that PpNSE5 either links two antiparallel SMC5/6 complexes or binds one SMC5/6 in elbow-bent conformation, the later model being consistent with the role of NSE5/NSE6 dimer as SMC5/6 loading factor to DNA lesions. In addition, we generated the P.patens Ppnse5KO1 mutant line with an N-terminally truncated version of PpNSE5, which exhibited DNA repair defects while keeping a normal number of rDNA repeats. As the first 71 amino acids of PpNSE5 are required for PpSMC6 binding, our results suggest the role of PpNSE5-PpSMC6 interaction in SMC5/6 loading to DNA lesions.

Assessing Transcriptomic Responses to Oxidative Stress: Contrasting Wild-Type Arabidopsis Seedlings with dss1(I) and dss1(V) Gene Knockout Mutants.

Oxidative stress represents a critical facet of the array of abiotic stresses affecting crop growth and yield. In this paper, we investigated the potential differences in the functions of two highly homologous Arabidopsis DSS1 proteins in terms of maintaining genome integrity and response to oxidative stress. In the context of homologous recombination (HR), it was shown that overexpressing AtDSS1(I) using a functional complementation test increases the resistance of the Δdss1 mutant of Ustilago maydis to genotoxic agents. This indicates its conserved role in DNA repair via HR. To investigate the global transcriptome changes occurring in dss1 plant mutant lines, gene expression analysis was conducted using Illumina RNA sequencing technology. Individual RNA libraries were constructed from three total RNA samples isolated from dss1(I), dss1(V), and wild-type (WT) plants under hydrogen peroxide-induced stress. RNA-Seq data analysis and real-time PCR identification revealed major changes in gene expression between mutant lines and WT, while the dss1(I) and dss1(V) mutant lines exhibited analogous transcription profiles. The Kyoto Encyclopedia of Genes and Genomes enrichment analysis revealed significantly enriched metabolic pathways. Notably, genes associated with HR were upregulated in dss1 mutants compared to the WT. Otherwise, genes of the metabolic pathway responsible for the synthesis of secondary metabolites were downregulated in both dss1 mutant lines. These findings highlight the importance of understanding the molecular mechanisms of plant responses to oxidative stress.

ZmNF-YA1 Contributes to Maize Thermotolerance by Regulating Heat Shock Response.

Zea mays (maize) is a staple food, feed, and industrial crop. Heat stress is one of the major stresses affecting maize production and is usually accompanied by other stresses, such as drought. Our previous study identified a heterotrimer complex, ZmNF-YA1-YB16-YC17, in maize. ZmNF-YA1 and ZmNF-YB16 were positive regulators of the drought stress response and were involved in maize root development. In this study, we investigated whether ZmNF-YA1 confers heat stress tolerance in maize. The nf-ya1 mutant and overexpression lines were used to test the role of ZmNF-YA1 in maize thermotolerance. The nf-ya1 mutant was more temperature-sensitive than the wild-type (WT), while the ZmNF-YA1 overexpression lines showed a thermotolerant phenotype. Higher malondialdehyde (MDA) content and reactive oxygen species (ROS) accumulation were observed in the mutant, followed by WT and overexpression lines after heat stress treatment, while an opposite trend was observed for chlorophyll content. RNA-seq was used to analyze transcriptome changes in nf-ya1 and its wild-type control W22 in response to heat stress. Based on their expression profiles, the heat stress response-related differentially expressed genes (DEGs) in nf-ya1 compared to WT were grouped into seven clusters via k-means clustering. Gene Ontology (GO) enrichment analysis of the DEGs in different clades was performed to elucidate the roles of ZmNF-YA1-mediated transcriptional regulation and their contribution to maize thermotolerance. The loss function of ZmNF-YA1 led to the failure induction of DEGs in GO terms of protein refolding, protein stabilization, and GO terms for various stress responses. Thus, the contribution of ZmNF-YA1 to protein stabilization, refolding, and regulation of abscisic acid (ABA), ROS, and heat/temperature signaling may be the major reason why ZmNF-YA1 overexpression enhanced heat tolerance, and the mutant showed a heat-sensitive phenotype.

Genotyping-by-Sequencing Analysis Reveals Associations between Agronomic and Oil Traits in Gamma Ray-Derived Mutant Rapeseed (Brassica napus L.).

Rapeseed (Brassica napus L.) holds significant commercial value as one of the leading oil crops, with its agronomic features and oil quality being crucial determinants. In this investigation, 73,226 single nucleotide polymorphisms (SNPs) across 95 rapeseed mutant lines induced by gamma rays, alongside the original cultivar ('Tamra'), using genotyping-by-sequencing (GBS) analysis were examined. This study encompassed gene ontology (GO) analysis and a genomewide association study (GWAS), thereby concentrating on agronomic traits (e.g., plant height, ear length, thousand-seed weight, and seed yield) and oil traits (including fatty acid composition and crude fat content). The GO analysis unveiled a multitude of genes with SNP variations associated with cellular processes, intracellular anatomical structures, and organic cyclic compound binding. Through GWAS, we detected 320 significant SNPs linked to both agronomic (104 SNPs) and oil traits (216 SNPs). Notably, two novel candidate genes, Bna.A05p02350D (SFGH) and Bna.C02p22490D (MDN1), are implicated in thousand-seed weight regulation. Additionally, Bna.C03p14350D (EXO70) and Bna.A09p05630D (PI4Kα1) emerged as novel candidate genes associated with erucic acid and crude fat content, respectively. These findings carry implications for identifying superior genotypes for the development of new cultivars. Association studies offer a cost-effective means of screening mutants and selecting elite rapeseed breeding lines, thereby enhancing the commercial viability of this pivotal oil crop.

Abolished clustering of MeCP2T158M can be partially reverted with small molecules

Rett syndrome (OMIM 312750) is a rare neurodevelopmental disorder caused by de novo mutations in the Methyl-CpG Binding Protein 2 (MeCP2) gene located on the X-chromosome, typically affecting girls. Rett syndrome symptoms, characterized by microcephaly and lack of motor coordination, first appear between 6 to 18 months of age. The disease continues to progress until adulthood at which point it reaches a stationary phase. Currently, available therapy for Rett Syndrome is only symptomatic. More than 800 mutations causing Rett syndrome have been described, the most common being T158M (9% prevalence) located in the Methyl-Binding Domain (MBD) of MeCP2. Due to its importance for DNA binding through recognition of methylated CpG, mutations in the MBD have a significant impact on the stability and function of MeCP2. MeCP2 is a nuclear protein and accumulates in liquid–liquid phase condensates visualized as speckles in NIH3T3 cells by microscopy. This speckled pattern is lost with MeCP2 mutations in the MBD such as T158M. We developed a high content phenotypic assay, detecting fluorescent MeCP2 speckles in NIH3T3 cells. The assay allows the identification of small molecules that stabilize MeCP2-T158M and phenotypically rescue speckle formation. To validate the assay, a collection of 3572 drugs was screened, including FDA-approved drugs, compounds in clinical trials and biologically annotated tool compounds. 18 hits showed at least 25% rescue of speckles in the mutant cell line while not affecting wild-type MeCP2 speckles. Primary hits were confirmed in a dose response assay, a thermal shift assay with recombinant MeCP2 and by testing the MeCP2 expression levels. One class of identified hits represents histone deacetylase inhibitors (HDACis) showing 25% speckle rescue of mutant MeCP2 without toxicity. This screening strategy can be expanded to additional compound libraries and will support novel drug discovery.

Functional divergence of FTL9 and FTL10 in flowering control in rice

BackgroundFloral transition in cereals is a critical phenomenon influenced by exogenous and endogenous signals, determining crop yield and reproduction. Flowering Locus T-like (FT-like) genes encode a mobile florigen, the main signaling molecule for flowering.ResultsIn this study, we characterized two FT-like genes, FTL9 and FTL10, to study their functional diversity in flowering control in rice. We compared independent mutant lines of ftl10 with WT and observed negligible differences in the flowering phenotype, or agronomic traits implying potentially redundant roles of FTL10 loss-of-function in flowering control in rice. Nevertheless, we found that overexpression of FTL10, but not FTL9, substantially accelerated flowering, indicating the flowering-promoting role of FTL10 and the divergent functions between FTL9 and FTL10 in flowering. Besides flowering, additive agronomic roles were observed for FTL10-OE regulating the number of effective panicles per plant, the number of primary branches per panicle, and spikelets per panicle without regulating seed size. Mechanistically, our Y2H and BiFC analyses demonstrate that FTL10, in contrast to FTL9, can interact with FD1 and GF14c, forming a flowering activation complex and thereby regulating flowering.ConclusionAltogether, our results elucidate the regulatory roles of FTL9 and FTL10 in flowering control, unveiling the molecular basis of functional divergence between FTL10 and FTL9, which provides mechanistic insights into shaping the dynamics of flowering time regulation in rice.

Disruption of recombination machinery alters the mutational landscape in plant organellar genomes.

Land plant organellar genomes have extremely low rates of point mutation yet also experience high rates of recombination and genome instability. Characterizing the molecular machinery responsible for these patterns is critical for understanding the evolution of these genomes. While much progress has been made towards understanding recombination activity in land plant organellar genomes, the relationship between recombination pathways and point mutation rates remains uncertain. The organellar targeted mutS homolog MSH1 has previously been shown to suppress point mutations as well as non-allelic recombination between short repeats in Arabidopsis thaliana. We therefore implemented high-fidelity Duplex Sequencing to test if other genes that function in recombination and maintenance of genome stability also affect point mutation rates. We found small to moderate increases in the frequency of single nucleotide variants (SNVs) and indels in mitochondrial and/or plastid genomes of A. thaliana mutant lines lacking radA, recA1, or recA3. In contrast, osb2 and why2 mutants did not exhibit an increase in point mutations compared to wild type (WT) controls. In addition, we analyzed the distribution of SNVs in previously generated Duplex Sequencing data from A. thaliana organellar genomes and found unexpected strand asymmetries and large effects of flanking nucleotides on mutation rates in WT plants and msh1 mutants. Finally, using long-read Oxford Nanopore sequencing, we characterized structural variants in organellar genomes of the mutant lines and show that different short repeat sequences become recombinationally active in different mutant backgrounds. Together, these complementary sequencing approaches shed light on how recombination may impact the extraordinarily low point mutation rates in plant organellar genomes.