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International Journal of Molecular... Nov 2022Water shortages caused by climate change seriously threaten the survival and production of plants and are also one of the major environmental pressures faced by plants....
Water shortages caused by climate change seriously threaten the survival and production of plants and are also one of the major environmental pressures faced by plants. DORN1 was the first identified purinoceptor for the plant response to extracellular ATP. It has been established that DORN1 could play key roles in a series of biological activities in plants. However, the biological roles of DORN1 and the mechanism remain unclear under drought stress conditions in plants. Here, DORN1 was targeted for knockout by using the CRISPR/Cas 9 system. It was found that the loss function of DORN1 resulted in a significant decrease in the effective quantum yield of PSII [Y(II)], the photochemical quenching coefficient (qP), and the rate of photosynthetic electron transport through PSII (ETR), which reflected plants' photochemical efficiency. Whereas Y values showed obvious enhancement under drought stress conditions. Further experimental results showed that the Y, q, and ETR, which reflect plants' photochemical efficiency, increased significantly with CaCl treatment. These results indicated that the drought tolerance of the mutant was decreased, and the exogenous application of calcium ions could effectively promote the drought tolerance of the mutant. Transpiration loss controlled by stomata is closely related to drought tolerance, further, we examined the transpirational water loss in and found that it was greater than wild-type (WT). Besides, the mutant's stomatal aperture significantly increased compared with the WT and the stomata of mutant plants tend to close after CaCl treatment. Taken together, our results show that DORN1 plays a key role in drought stress tolerance in plants, which may depend on calcium and calcium-related signaling pathways.
Topics: Droughts; Calcium; Calcium Chloride; Photosynthesis; Water
PubMed: 36430696
DOI: 10.3390/ijms232214213 -
Genetics Feb 2020Plants integrate internal and external signals to finely coordinate growth and defense for maximal fitness within a complex environment. A common model suggests that...
Plants integrate internal and external signals to finely coordinate growth and defense for maximal fitness within a complex environment. A common model suggests that growth and defense show a trade-offs relationship driven by energy costs. However, recent studies suggest that the coordination of growth and defense likely involves more conditional and intricate connections than implied by the trade-off model. To explore how a transcription factor (TF) network may coordinate growth and defense, we used a high-throughput phenotyping approach to measure growth and flowering in a set of single and pairwise mutants previously linked to the aliphatic glucosinolate (GLS) defense pathway. Supporting a link between growth and defense, 17 of the 20 tested defense-associated TFs significantly influenced plant growth and/or flowering time. The TFs' effects were conditional upon the environment and age of the plant, and more critically varied across the growth and defense phenotypes for a given genotype. In support of the coordination model of growth and defense, the TF mutant's effects on short-chain aliphatic GLS and growth did not display a simple correlation. We propose that large TF networks integrate internal and external signals and separately modulate growth and the accumulation of the defensive aliphatic GLS.
Topics: Arabidopsis; Arabidopsis Proteins; Epistasis, Genetic; Fatty Acids; Gene Expression Regulation, Plant; Genotype; Glucosinolates; Mutation; Phenotype; Transcription Factors
PubMed: 31852726
DOI: 10.1534/genetics.119.302996 -
Methods in Molecular Biology (Clifton,... 2021Complex genetic interactions occur when mutant alleles of multiple genes combine to elicit an unexpected phenotype, which could not be predicted given the expectation...
Complex genetic interactions occur when mutant alleles of multiple genes combine to elicit an unexpected phenotype, which could not be predicted given the expectation based on the combination of phenotypes associated with individual mutant alleles. Trigenic Synthetic Genetic Array (τ-SGA) methodology was developed for the systematic analysis of complex interactions involving combinations of three gene perturbations. With a series of replica pinning steps of the τ-SGA procedure, haploid triple mutants are constructed through automated mating and meiotic recombination. For example, a double-mutant query strain carrying two mutant alleles of interest, such as a deletion allele of a nonessential gene and a conditional temperature-sensitive allele of an essential gene, is crossed to an input array of yeast mutants, such as the diagnostic array set of ~1200 mutants, to generate an output array of triple mutants. The colony-size measurements of the resulting triple mutants are used to estimate cellular fitness and quantify trigenic interactions by incorporating corresponding single- and double-mutant fitness estimates. Trigenic interaction networks can be further analyzed for functional modules using various clustering and enrichment analysis tools. Complex genetic interactions are rich in functional information and provide insight into the genotype-to-phenotype relationship, genome size, and speciation.
Topics: Alleles; Epistasis, Genetic; Gene Expression Regulation, Fungal; Gene Regulatory Networks; Genes, Essential; Genes, Fungal; Genes, Synthetic; Genotype; Haploidy; Mutation; Oligonucleotide Array Sequence Analysis; Phenotype; Saccharomyces cerevisiae
PubMed: 33733368
DOI: 10.1007/978-1-0716-0947-7_23 -
Cell & Bioscience 2020Epidermal growth factor receptor (EGFR) has emerged as an important therapeutic target. Overexpression of EGFR is frequently observed in hepatocellular carcinoma (HCC)...
BACKGROUND
Epidermal growth factor receptor (EGFR) has emerged as an important therapeutic target. Overexpression of EGFR is frequently observed in hepatocellular carcinoma (HCC) and EGFR activation has been proven to be a potential determinant of primary resistance of HCC cells to sorafenib. In our previous study, we found 13 missense mutations in EGFR exon 19-23 from hepatocellular carcinoma (HCC) tissues, but the functions of these mutations have not been determined. This study aims to determine the kinase activity and sensitivity to erlotinib, a 1st-generation EGFR-tyrosine kinase inhibitor (TKI), of seven HCC-derived mutants (K757E, N808S, R831C, V897A, P937L, T940A, and M947T).
RESULTS
Using transduction of pBabe-puro retroviral vector with or without EGFR, we constructed and determined the function of EGFRs in NIH-3T3 cells stably harboring each of the seven mutants, as well as the erlotinib-sensitive L858R-mutant, the erlotinib-resistant T790M-mutant, and EGFR wild type (WT). Our results indicate that the seven mutants are functioning, EGF-dependent, EGFRs. Cells harboring six of the seven mutants could generate some level of EGFR phosphorylation in the absence of EGF, indicating some constitutive kinase activity, but all of the seven mutants remain primarily EGF-dependent. Our results demonstrate that erlotinib induces differential degree of apoptosis and autophagy among cells harboring different EGFRs: complete apoptosis and autophagy (cleavage of both caspase-3 and PARP, and marked LC3-II increment) in L858R-mutant; partial apoptosis and autophagy (only cleavage of caspase-3, and moderate LC3-II increment) in WT and HCC-derived mutants; and no apoptosis and minimal autophagy (no cleavage of caspase-3 and PARP, and minimal LC3-II increment) in T790M-mutant. The seven HCC-derived mutants are erlotinib-resistant, as treatment with erlotinib up to high concentration could only induce partial inhibition of EGFR phosphorylation, partial or no inhibition of AKT and ERK phosphorylation, and partial apoptosis and autophagy.
CONCLUSION
The seven HCC-derived EGFR mutants in this study are functioning, EGF-dependent, and erlotinib-resistant. Erlotinib induces differential degree of apoptosis and autophagy among cells harboring different EGFRs. The degree of inhibition of EGFR phosphorylation by erlotinib is the determining factor for the degree of apoptosis and autophagy amongst cells harboring EGFR mutants. This study paves the way for further investigation into the sensitivity of these HCC-derived mutants to the 3rd-generation irreversible EGFR-TKI, osimertinib.
PubMed: 32190291
DOI: 10.1186/s13578-020-00407-1 -
Canadian Journal of Microbiology Nov 2020Brewer's yeast has been widely used in the food industry, and the autolysates thereof are increasingly being studied for their valuable nutritional compositions. Yeast...
Brewer's yeast has been widely used in the food industry, and the autolysates thereof are increasingly being studied for their valuable nutritional compositions. Yeast autolysis is most affected by medium composition and temperature. In this study, a thermosensitive autolytic brewer's yeast P-510 was obtained with atmospheric and room temperature plasma mutagenesis plus 5-bromo-chloro-3-indolyl phosphate screening. The mutant rapidly autolyzed at 37 °C and the autolysates contained more active components and showed higher antioxidant activities compared with that of the parental strain, which indicated that the mutant's autolysates can potentially be used as functional food and nutritional ingredients. Transcriptomic analysis of the mutant and parental strains at 28 and 37 °C suggested that thermosensitive autolysis of P-510 was probably caused by mitochondrial disfunction, glycogen metabolic flux of glycolysis and pentose phosphate pathway disorder, as well as hexose transport inhibition. The results revealed the important role of mitochondrial metabolism and glycogen utilization regulation in heat stress response of yeast.
Topics: Antioxidants; Gene Expression Profiling; Heat-Shock Response; Mutagenesis; Mutation; Saccharomyces cerevisiae; Temperature
PubMed: 32619357
DOI: 10.1139/cjm-2019-0456 -
Microorganisms Sep 2022is a major cause of skin/soft tissue infections and more serious infections in humans. The species usually requires the importation of proline to be able to survive....
is a major cause of skin/soft tissue infections and more serious infections in humans. The species usually requires the importation of proline to be able to survive. Previous work has shown that single mutations in genes that encode for proline transporters affect the ability of to survive in vitro and in vivo. To better understand proline transport in , double and triple gene mutant strains were created that targeted the , , and genes. Single gene mutants had some effect on proline transport, whereas double mutants exhibited significantly lower proline transport. An triple gene mutant displayed the lowest proline transport under low- and high-affinity conditions. To assess growth differences caused by the mutations, the same mutants were grown in brain heart infusion (BHI) broth and defined staphylococcal medium (DSM) with various concentrations of proline. The triple mutant did not grow in DSM with a low concentration of proline and grew poorly in both DSM with a high proline concentration and BHI broth. These results show that has multiple mechanisms to import proline into the cell and knocking out three of the main proline transporters significantly hinders growth.
PubMed: 36296165
DOI: 10.3390/microorganisms10101888 -
Plant Direct Jul 2023The developmental genetics of reproductive structure control in maize must consider both the staminate florets of the tassel and the pistillate florets of the ear...
The developmental genetics of reproductive structure control in maize must consider both the staminate florets of the tassel and the pistillate florets of the ear synflorescences. Pistil abortion takes place in the tassel florets, and stamen arrest is affected in ear florets to give rise to the monoecious nature of maize. Gibberellin (GA) deficiency results in increased tillering, a dwarfed plant syndrome, and the retention of anthers in the ear florets of maize. The mutant results in suppression of silks in the ear. We demonstrate in this study that jasmonic acid (JA) and GA act independently and show additive phenotypes resulting in androecious double mutant plants. The persistence of pistils in the tassel can be induced by multiple mechanisms, including JA deficiency, GA excess, genetic control of floral determinacy, and organ identity. The mutant can suppress both silks in the ear and the silks in the tassel of JA-deficient and AP2 transcription factor mutants. We previously demonstrated that GA production was required for brassinosteroid (BR) deficiency to affect persistence of pistils in the tassel. We find that BR deficiency affects pistil persistence by an independent mechanism from the mutant and JA pathway. The mutant did not prevent the formation of pistils in the tassel by in double mutants. In addition, we demonstrate that there is more to the mutant than just a suppression of pistil growth. We document novel phenotypes of mutants including weakly penetrant ear fasciation and anther persistence in the ear florets. Thus, the JA/AP2 mechanism of pistil retention in the tassel and silk growth in the ear are similarly sensitive to loss of the SILKLESS1 protein, while the BR/GA mechanism is not.
PubMed: 37440932
DOI: 10.1002/pld3.501 -
Plant Diversity Feb 2020Carotenoids are important pigments in photosynthetic organisms where they play essential roles in photoreception and photoprotection. is a unicellular green alga that...
Carotenoids are important pigments in photosynthetic organisms where they play essential roles in photoreception and photoprotection. is a unicellular green alga that is able to accumulate high amounts of ketocarotenoids including astaxanthin, canthaxanthin and ketolutein when growing heterotrophically or mixotrophically with glucose as a carbon source. Here we elucidate the ketocarotenoid biosynthesis pathway in by analyzing five algal mutants. The mutants were shown to have a single nucleotide insertion or substitution in β-carotene ketolase () gene 1, which resulted in a lack of ketocarotenoid production in Cz-bkt1-1, and decreased ketocarotenoid content in the other four mutants. These mutants accumulated much higher amounts of non-ketocarotenoids (β-carotene, zeaxanthin and lutein). Interestingly, the Cz-bkt1-5 mutant synthesized 2-fold the ketolutein and only 1/30 of the canthaxanthin and astaxanthin as its parent strain, suggesting that the mutated BKT1 exhibits much higher activity in catalyzing lutein to ketolutein but lower activity in ketolating β-carotene and zeaxanthin. Mutant and WT 2 gene sequences did not differ. Taken together, we conclude that 1 is the key gene involved in ketocarotenoid biosynthesis in . Our study provides insight into the biosynthesis of ketocarotenoids in green algae. Furthermore, Cz-bkt1 mutants may serve as a natural source for the production of zeaxanthin, lutein, and β-carotene.
PubMed: 32140638
DOI: 10.1016/j.pld.2019.11.001 -
The European Journal of Neuroscience May 2022Affective behaviours and mental health are profoundly affected by disturbances in circadian rhythms. Casein kinase 1 epsilon (CSNK1E) is a core component of the...
Affective behaviours and mental health are profoundly affected by disturbances in circadian rhythms. Casein kinase 1 epsilon (CSNK1E) is a core component of the circadian clock. Mice with tau or null mutation of this gene have shortened and lengthened circadian period respectively. Here, we examined anxiety-like, fear, and despair behaviours in both male and female mice of these two different mutants. Compared with wild-type mice, we found reductions in fear and anxiety-like behaviours in both mutant lines and in both sexes, with the tau mutants exhibiting the greatest phenotypic changes. However, the behavioural despair had distinct phenotypic patterns, with markedly less behavioural despair in female null mutants, but not in tau mutants of either sex. To determine whether abnormal light entrainment of tau mutants to 24-h light-dark cycles contributes to these phenotypic differences, we also examined these behaviours in tau mutants on a 20-h light-dark cycle close to their endogenous circadian period. The normalized entrainment restored more wild-type-like behaviours for fear and anxiety, but it induced behavioural despair in tau mutant females. These data show that both mutations of Csnk1e broadly affect fear and anxiety-like behaviours, while the effects on behavioural despair vary with genetics, photoperiod, and sex, suggesting that the mechanisms by which Csnk1e affects fear and anxiety-like behaviours may be similar, but distinct from those affecting behavioural despair. Our study also provides experimental evidence in support of the hypothesis of beneficial outcomes from properly entrained circadian rhythms in terms of the anxiety-like and fear behaviours.
Topics: Animals; Casein Kinase 1 epsilon; Circadian Clocks; Circadian Rhythm; Female; Male; Mice; Motor Activity; Photoperiod
PubMed: 34514665
DOI: 10.1111/ejn.15456 -
Journal of Bacteriology Nov 2021The alphaproteobacterium Sinorhizobium meliloti secretes two acidic exopolysaccharides (EPSs), succinoglycan (EPSI) and galactoglucan (EPSII), which differentially...
The alphaproteobacterium Sinorhizobium meliloti secretes two acidic exopolysaccharides (EPSs), succinoglycan (EPSI) and galactoglucan (EPSII), which differentially enable it to adapt to a changing environment. Succinoglycan is essential for invasion of plant hosts and, thus, for the formation of nitrogen-fixing root nodules. Galactoglucan is critical for population-based behaviors such as swarming and biofilm formation and can facilitate invasion in the absence of succinoglycan on some host plants. The biosynthesis of galactoglucan is not as completely understood as that of succinoglycan. We devised a pipeline to identify putative pyruvyltransferase and acetyltransferase genes, construct genomic deletions in strains engineered to produce either succinoglycan or galactoglucan, and analyze EPS from mutant bacterial strains. EPS samples were examined by C cross-polarization magic-angle spinning (CPMAS) solid-state nuclear magnetic resonance (NMR). CPMAS NMR is uniquely suited to defining chemical composition in complex samples and enables the detection and quantification of distinct EPS functional groups. Galactoglucan was isolated from mutant strains with deletions in five candidate acyl/acetyltransferase genes (, , , , and ) and a putative pyruvyltransferase ( or ). Most samples were similar in composition to wild-type EPSII by CPMAS NMR analysis. However, galactoglucan produced from a strain lacking exhibited a significant reduction in pyruvylation. Pyruvylation was restored through the ectopic expression of plasmid-borne . Our work has thus identified WgaE as a galactoglucan pyruvyltransferase. This exemplifies how the systematic combination of genetic analyses and solid-state NMR detection is a rapid means to identify genes responsible for modification of rhizobial exopolysaccharides. Nitrogen-fixing bacteria are crucial for geochemical cycles and global nitrogen nutrition. Symbioses between legumes and rhizobial bacteria establish root nodules, where bacteria convert dinitrogen to ammonia for plant utilization. Secreted exopolysaccharides (EPSs) produced by Sinorhizobium meliloti (succinoglycan and galactoglucan) play important roles in soil and plant environments. The biosynthesis of galactoglucan is not as well characterized as that of succinoglycan. We employed solid-state nuclear magnetic resonance (NMR) to examine intact EPS from wild-type and mutant S. meliloti strains. NMR analysis of EPS isolated from a gene mutant revealed a novel pyruvyltransferase that modifies galactoglucan. Few EPS pyruvyltransferases have been characterized. Our work provides insight into the biosynthesis of an important S. meliloti EPS and expands the knowledge of enzymes that modify polysaccharides.
Topics: Bacterial Proteins; Galactans; Gene Expression Regulation, Bacterial; Gene Expression Regulation, Enzymologic; Glucans; Humans; Magnetic Resonance Spectroscopy; Mutation; Polysaccharides, Bacterial; Sinorhizobium meliloti; Transferases
PubMed: 34606371
DOI: 10.1128/JB.00403-21