Temperature sensitivity and tolerance play a key role in plant survival and production.Perennial ryegrass(Lolium perenne L.),widely cultivated in cool-season for forage supply and turfgrass,is extremely susceptible to...Temperature sensitivity and tolerance play a key role in plant survival and production.Perennial ryegrass(Lolium perenne L.),widely cultivated in cool-season for forage supply and turfgrass,is extremely susceptible to high temperatures,therefore serving as an excellent grass for dissecting the genomic and genetic basis of high-temperature adaptation.In this study,expression analysis revealed that LpHsfA2,an important gene associated with high-temperature tolerance in perennial ryegrass,is rapidly and substantially induced under heat stress.Additionally,heat-tolerant varieties consistently display elevated expression levels of LpHsfA2 compared with heat-sensitive ones.Comparative haplotype analysis of the LpHsfA2 promoter indicated an uneven distribution of two haplotypes(HsfA2^(Hap1) and HsfA2^(Hap2)) across varieties with differing heat tolerance.Specifically,the HsfA2^(Hap1) allele is predominantly present in heat-tolerant varieties,while the HsfA2^(Hap2) allele exhibits the opposite pattern.Overexpression of LpHsfA2 confers enhanced thermotolerance,whereas silencing of LpHsfA2 compromises heat tolerance.Furthermore,LpHsfA2 orchestrates its protective effects by directly binding to the promoters of LpHSP18.2 and LpAPX1 to activate their expression,preventing the non-specific misfolding of intracellular protein and the accumulation of reactive oxygen species in cells.Additionally,LpHsf A4 and LpHsf A5 were shown to engage directly with the promoter of LpHsfA2,upregulating its expression as well as the expression of LpHSP18.2 and LpAPX1,thus contributing to enhanced heat tolerance.Markedly,LpHsfA2 possesses autoregulatory ability by directly binding to its own promoter to modulate the self-transcription.Based on these findings,we propose a model for modulating the thermotolerance of perennial ryegrass by precisely regulating the expression of LpHsfA2.Collectively,these findings provide a scientific basis for the development of thermotolerant perennial ryegrass cultivars.展开更多
Leaf senescence is driven by the expression of senescence-associated genes(SAGs).Development-specific genes often undergo DNA demethylation in their promoter and other regions,which regulates gene expression.Whether a...Leaf senescence is driven by the expression of senescence-associated genes(SAGs).Development-specific genes often undergo DNA demethylation in their promoter and other regions,which regulates gene expression.Whether and how DNA demethylation regulates the expression of SAGs and thus leaf senescence remain elusive.Whole-genome bisulfite sequencing(WGBS)analyses of wild-type(WT)and demeter-like 3(dml3)Arabidopsis leaves at three developmental stages revealed hypermethylation during leaf senescence in dml3 compared with WT,and 20556 differentially methylated regions(DMRs)were identified by comparing the methylomes of dml3 and WT in the CG,CHG,and CHH contexts.Furthermore,we identified that 335 DMR-associated genes(DMGs),such as NAC016 and SEN1,are upregulated during leaf senescence,and found an inverse correlation between the DNA methylation levels(especially in the promoter regions)and the transcript abundances of the related SAGs in WT.In contrast,in dml3 the promoters of SAGs were hypermethylated and their transcript levels were remarkably reduced,and leaf senescence was significantly delayed.Collectively,our study unraveled a novel epigenetic regulatory mechanism underlying leaf senescence in which DML3 is expressed at the onset of and during senescence to demethylate promoter,gene body or 3'UTR regions to activate a set of SAGs.展开更多
Clustered regularly interspaced palindromic repeats(CRISPR)/CRISPR-associated protein 9(Cas9)system has been widely used for precise gene editing in plants.However,simultaneous gene editing of multiple homoeoalleles r...Clustered regularly interspaced palindromic repeats(CRISPR)/CRISPR-associated protein 9(Cas9)system has been widely used for precise gene editing in plants.However,simultaneous gene editing of multiple homoeoalleles remains challenging,especially in self-incompatible polyploid plants.Here,we simultaneously introduced targeted mutations in all three homoeoalleles of two genes in the self-incompatible allohexaploid tall fescue,using both CRISPR/Cas9 and LbCas12a(LbCpf1)systems.Loss-of-function mutants of FaPDS exhibited albino leaves,while knockout of FaHSP17.9 resulted in impaired heat resistance in T0 generation of tall fescue.Moreover,these mutations were inheritable.Our findings demonstrate the feasibility of generating loss-of-function mutants in T0 generation polyploid perennial grasses using CRISPR/Cas systems.展开更多
基金supported by the Fundamental Research Funds for the Central Universities (YD9100002013)the National Key Research and Development Program of China (2021YFC2300300)+1 种基金the National Natural Science Foundation of China (32070132)the Strategic Priority Research Program of the Chinese Academy of Sciences (XDB29020000)
基金supported by the National Key R&D Program of China(2022YFF1003200)the National Natural Science Foundation of China(NSFC)(Grant Nos.32001394,32102431 and 32101430)+2 种基金the Science&Technology Specific Projects in Agricultural High-tech Industrial Demonstration Area of the Yellow River Delta(Grant No.2022SZX13)the Strategic Priority Research Program of Chinese Academy of Sciences(Grant No.XDA26050201)the Major Science and Technology Innovation Project of Shandong Province(2022LZGC018)。
文摘Temperature sensitivity and tolerance play a key role in plant survival and production.Perennial ryegrass(Lolium perenne L.),widely cultivated in cool-season for forage supply and turfgrass,is extremely susceptible to high temperatures,therefore serving as an excellent grass for dissecting the genomic and genetic basis of high-temperature adaptation.In this study,expression analysis revealed that LpHsfA2,an important gene associated with high-temperature tolerance in perennial ryegrass,is rapidly and substantially induced under heat stress.Additionally,heat-tolerant varieties consistently display elevated expression levels of LpHsfA2 compared with heat-sensitive ones.Comparative haplotype analysis of the LpHsfA2 promoter indicated an uneven distribution of two haplotypes(HsfA2^(Hap1) and HsfA2^(Hap2)) across varieties with differing heat tolerance.Specifically,the HsfA2^(Hap1) allele is predominantly present in heat-tolerant varieties,while the HsfA2^(Hap2) allele exhibits the opposite pattern.Overexpression of LpHsfA2 confers enhanced thermotolerance,whereas silencing of LpHsfA2 compromises heat tolerance.Furthermore,LpHsfA2 orchestrates its protective effects by directly binding to the promoters of LpHSP18.2 and LpAPX1 to activate their expression,preventing the non-specific misfolding of intracellular protein and the accumulation of reactive oxygen species in cells.Additionally,LpHsf A4 and LpHsf A5 were shown to engage directly with the promoter of LpHsfA2,upregulating its expression as well as the expression of LpHSP18.2 and LpAPX1,thus contributing to enhanced heat tolerance.Markedly,LpHsfA2 possesses autoregulatory ability by directly binding to its own promoter to modulate the self-transcription.Based on these findings,we propose a model for modulating the thermotolerance of perennial ryegrass by precisely regulating the expression of LpHsfA2.Collectively,these findings provide a scientific basis for the development of thermotolerant perennial ryegrass cultivars.
基金This work was supported by the National Key Research and Development Program of China(grant no.2019YFD1000300,to L.Chen)by Cornell University 1843351(to S.G.).
文摘Leaf senescence is driven by the expression of senescence-associated genes(SAGs).Development-specific genes often undergo DNA demethylation in their promoter and other regions,which regulates gene expression.Whether and how DNA demethylation regulates the expression of SAGs and thus leaf senescence remain elusive.Whole-genome bisulfite sequencing(WGBS)analyses of wild-type(WT)and demeter-like 3(dml3)Arabidopsis leaves at three developmental stages revealed hypermethylation during leaf senescence in dml3 compared with WT,and 20556 differentially methylated regions(DMRs)were identified by comparing the methylomes of dml3 and WT in the CG,CHG,and CHH contexts.Furthermore,we identified that 335 DMR-associated genes(DMGs),such as NAC016 and SEN1,are upregulated during leaf senescence,and found an inverse correlation between the DNA methylation levels(especially in the promoter regions)and the transcript abundances of the related SAGs in WT.In contrast,in dml3 the promoters of SAGs were hypermethylated and their transcript levels were remarkably reduced,and leaf senescence was significantly delayed.Collectively,our study unraveled a novel epigenetic regulatory mechanism underlying leaf senescence in which DML3 is expressed at the onset of and during senescence to demethylate promoter,gene body or 3'UTR regions to activate a set of SAGs.
基金This work was supported by the National Natural Science Foundation of China(31772349,31672482,and 31401915)the Major Science and Technology Innovation Project of Shandong Province(2019JZZY010726).
文摘Clustered regularly interspaced palindromic repeats(CRISPR)/CRISPR-associated protein 9(Cas9)system has been widely used for precise gene editing in plants.However,simultaneous gene editing of multiple homoeoalleles remains challenging,especially in self-incompatible polyploid plants.Here,we simultaneously introduced targeted mutations in all three homoeoalleles of two genes in the self-incompatible allohexaploid tall fescue,using both CRISPR/Cas9 and LbCas12a(LbCpf1)systems.Loss-of-function mutants of FaPDS exhibited albino leaves,while knockout of FaHSP17.9 resulted in impaired heat resistance in T0 generation of tall fescue.Moreover,these mutations were inheritable.Our findings demonstrate the feasibility of generating loss-of-function mutants in T0 generation polyploid perennial grasses using CRISPR/Cas systems.
