Abscisic acid(ABA)is a critical regulator of seed development and germination.β-glucosidases(BGs)have been suggested to be contributors to increased ABA content because they catalyze the hydrolysis of ABA-glucose est...Abscisic acid(ABA)is a critical regulator of seed development and germination.β-glucosidases(BGs)have been suggested to be contributors to increased ABA content because they catalyze the hydrolysis of ABA-glucose ester to release free ABA.However,whether BGs are involved in seed development is unclear.In this study,a candidate gene,CiBG1,in watermelon was selected for targeted mutagenesis via the CRISPR/Cas9 system.Seed size and weight were significantly reduced in the Clbg1-mutant watermelon lines,which was mainly attributed to decreased cell number resulting from decreased ABA levels.A transcriptome analysis showed that the expression of 1015 and 1429 unique genes was changed 10 and 18 days after pollination(DAP),respectively.Cytoskeleton-and cell cycle-related genes were enriched in the differentially expressed genes of wild type and Clbg1-mutant lines during seed development.Moreover,the expression of genes in the major signaling pathways of seed size control was also changed.In addition,seed germination was promoted in the Cibg1-mutant lines due to decreased ABA content.These results indicate that ClBG1 may be critical for watermelon seed size regulation and germination mainly through the modulation of ABA content and thereby the transcriptional regulation of cytoskeleton-,cell cycle-and signaling-related genes.Our results lay a foundation for dissecting the molecular mechanisms of controlling watermelon seed size,a key agricultural trait of significant economic importance.展开更多
NAC(NAM,ATAF1/2,and CUC2)transcription factors play important roles in fruit ripening and quality.The watermelon genome encodes 80 NAC genes,and 21 of these NAC genes are highly expressed in both the flesh and vascula...NAC(NAM,ATAF1/2,and CUC2)transcription factors play important roles in fruit ripening and quality.The watermelon genome encodes 80 NAC genes,and 21 of these NAC genes are highly expressed in both the flesh and vascular tissues.Among these genes,CINAC68 expression was signi fi cantly higher in flesh than in rind.However,the intrinsic regulatory mechanism of CINAC68 in fruit ripening and quality is still unknown.In this study,we found that ClNAC68 is a transcriptional repressor and that the repression domain is located in the C-terminus.Knockout of CINAC68 by the CRISPR-Cas9 system decreased the soluble solid content and sucrose accumulation in mutant flesh.Development was delayed,germination was inhibited,and the IAA content was signi ficantly decreased in mutant seeds.Transcriptome analysis showed that the invertase gene CUNV was the only gene involved in sucrose metabolism that was upregulated in mutant flesh,and expression of the indole-3-acetic acid-amido synthetase gene CIGH3.6 in the IAA signaling pathway was also induced in mutant seeds.EMSA and dual-luciferase assays showed that CINAC68 directly bound to the promoters of CUNV and CIGH3.6 to repress their expression.These results indicated that CINAC68 positively regulated sugar and IAA accumulation by repressing CUNV and CIGH3.6.Our findings provide new insights into the regulatory mechanisms by which NAC transcription factors affect fruit quality and seed development.展开更多
Although crop domestication has greatly aided human civilization,the sequential domestication and regulation of most quality traits remain poorly understood.Here,we report the stepwise selection and regulation of majo...Although crop domestication has greatly aided human civilization,the sequential domestication and regulation of most quality traits remain poorly understood.Here,we report the stepwise selection and regulation of major fruit quality traits that occurred during watermelon evolution.The levels of fruit cucurbitacins and flavonoids were negatively selected during speciation,whereas sugar and carotenoid contents were positively selected during domestication.Interestingly,fruit malic acid and citric acid showed the opposite selection trends during the improvement.We identified a novel gene cluster(CGC1,cucurbitacin gene cluster on chromosome 1)containing both regulatory and structural genes involved in cucurbitacin biosynthesis,which revealed a cascade of transcriptional regulation operating mechanisms.In the CGC1,an allele caused a single nucleotide change in Cl ERF1 binding sites(GCC-box)in the promoter of Cl Bh1,which resulted in reduced expression of Cl Bh1 and inhibition of cucurbitacin synthesis in cultivated watermelon.Functional analysis revealed that a rare insertion of 244 amino acids,which arose in C.amarus and became fixed in sweet watermelon,in Cl OSC(oxidosqualene cyclase)was critical for the negative selection of cucurbitacins during watermelon evolution.This research provides an important resource for metabolomics-assisted breeding in watermelon and for exploring metabolic pathway regulation mechanisms.展开更多
ThThe NAC transcription factor NONRIPENING(NOR)is a master regulator of climacteric fruit ripening.Melon(Cucumis melo L.)has climacteric and nonclimacteric fruit ripening varieties and is an ideal model to study fruit...ThThe NAC transcription factor NONRIPENING(NOR)is a master regulator of climacteric fruit ripening.Melon(Cucumis melo L.)has climacteric and nonclimacteric fruit ripening varieties and is an ideal model to study fruit ripening.Two natural CmNAC-NOR variants,the climacteric haplotype CmNAC-NOR^(S,N) and the non-climacteric haplotype CmNAC-NOR^(A,S),have effects on fruit ripening;however,their regulatory mechanisms have not been elucidated.Here,we report that a natural mutation in the transcriptional activation domain of CmNAC-NORS,Ncontributes to climacteric melon fruit ripening.CmNAC-NOR knockout in the climacteric-type melon cultivar“BYJH”completely inhibited fruit ripening,while ripening was delayed by 5-8 d in heterozygous cmnac-nor mutant fruits.CmN AC-NOR directly activated carotenoid,ethylene,and abscisic acid biosynthetic genes to promote fruit coloration and ripening.Furthermore,CmNAC-NOR mediated the transcription of the“CmNAC-NOR-CmNAC73-CmCWINV2”module to enhance flesh sweetness.The transcriptional activation activity of the climacteric haplotype CmNAC-NORS,Non these target genes was significantly higher than that of the nonclimacteric haplotype CmNAC-NOR^(A,S).Moreover,CmNAC-NORS,Ncomplementation fully rescued the non-ripening phenotype of the tomato(Solanum lycopersicum)cr-nor mutant,while CmNAC-NOR^(A,S) did not.Our results provide insight into the molecular mechanism of climacteric and non-climacteric fruit ripening in melon.展开更多
基金supported by the National Natural Science Foundation of China(NSFC Grant Nos.31701938,31930096,and 1902034)Collaborative Innovation Center of BAAFS(KJCX201907-2)+2 种基金Ministry of Agriculture and Rural Affairs of China(Grant No.CARS-25)Beijing Scholar Program(Grant No.BSP026)Guanxi Bagui Scholar Program(Grant No.2016A11).
文摘Abscisic acid(ABA)is a critical regulator of seed development and germination.β-glucosidases(BGs)have been suggested to be contributors to increased ABA content because they catalyze the hydrolysis of ABA-glucose ester to release free ABA.However,whether BGs are involved in seed development is unclear.In this study,a candidate gene,CiBG1,in watermelon was selected for targeted mutagenesis via the CRISPR/Cas9 system.Seed size and weight were significantly reduced in the Clbg1-mutant watermelon lines,which was mainly attributed to decreased cell number resulting from decreased ABA levels.A transcriptome analysis showed that the expression of 1015 and 1429 unique genes was changed 10 and 18 days after pollination(DAP),respectively.Cytoskeleton-and cell cycle-related genes were enriched in the differentially expressed genes of wild type and Clbg1-mutant lines during seed development.Moreover,the expression of genes in the major signaling pathways of seed size control was also changed.In addition,seed germination was promoted in the Cibg1-mutant lines due to decreased ABA content.These results indicate that ClBG1 may be critical for watermelon seed size regulation and germination mainly through the modulation of ABA content and thereby the transcriptional regulation of cytoskeleton-,cell cycle-and signaling-related genes.Our results lay a foundation for dissecting the molecular mechanisms of controlling watermelon seed size,a key agricultural trait of significant economic importance.
基金This work was financially supported by the Collaborative Innovation Center of BAAFS(KJCX201907-2)Beijing Science&Technology Program(D171100007617001)+3 种基金National Key R&D Program of China(2018YFD0100703)National Natural Science Foun dation of China(31930096 and 1902034)Beijing Natural Science Foundation(6204038 and 6202010)Beijing Scholar Program(BSP026 and YBSP019).
文摘NAC(NAM,ATAF1/2,and CUC2)transcription factors play important roles in fruit ripening and quality.The watermelon genome encodes 80 NAC genes,and 21 of these NAC genes are highly expressed in both the flesh and vascular tissues.Among these genes,CINAC68 expression was signi fi cantly higher in flesh than in rind.However,the intrinsic regulatory mechanism of CINAC68 in fruit ripening and quality is still unknown.In this study,we found that ClNAC68 is a transcriptional repressor and that the repression domain is located in the C-terminus.Knockout of CINAC68 by the CRISPR-Cas9 system decreased the soluble solid content and sucrose accumulation in mutant flesh.Development was delayed,germination was inhibited,and the IAA content was signi ficantly decreased in mutant seeds.Transcriptome analysis showed that the invertase gene CUNV was the only gene involved in sucrose metabolism that was upregulated in mutant flesh,and expression of the indole-3-acetic acid-amido synthetase gene CIGH3.6 in the IAA signaling pathway was also induced in mutant seeds.EMSA and dual-luciferase assays showed that CINAC68 directly bound to the promoters of CUNV and CIGH3.6 to repress their expression.These results indicated that CINAC68 positively regulated sugar and IAA accumulation by repressing CUNV and CIGH3.6.Our findings provide new insights into the regulatory mechanisms by which NAC transcription factors affect fruit quality and seed development.
基金supported by the Agricultural Science and Technology Innovation Program(CAAS-ASTIP-ZFRI-07)the National Key R&D Program of China(2018YFD0100704)+5 种基金the China Agriculture Research System(CARS-25-03)the National Natural Science Fund for Distinguished Young Scholars(31625021)the National Natural Science Foundation of China(31672178,31471893)the Hainan University Startup Fund KYQD(ZR)1866Project supported by Hainan Yazhou Bay Seed Laboratory(B21Y10901)the Natural Science Foundation of Hainan Province(322RC574)。
文摘Although crop domestication has greatly aided human civilization,the sequential domestication and regulation of most quality traits remain poorly understood.Here,we report the stepwise selection and regulation of major fruit quality traits that occurred during watermelon evolution.The levels of fruit cucurbitacins and flavonoids were negatively selected during speciation,whereas sugar and carotenoid contents were positively selected during domestication.Interestingly,fruit malic acid and citric acid showed the opposite selection trends during the improvement.We identified a novel gene cluster(CGC1,cucurbitacin gene cluster on chromosome 1)containing both regulatory and structural genes involved in cucurbitacin biosynthesis,which revealed a cascade of transcriptional regulation operating mechanisms.In the CGC1,an allele caused a single nucleotide change in Cl ERF1 binding sites(GCC-box)in the promoter of Cl Bh1,which resulted in reduced expression of Cl Bh1 and inhibition of cucurbitacin synthesis in cultivated watermelon.Functional analysis revealed that a rare insertion of 244 amino acids,which arose in C.amarus and became fixed in sweet watermelon,in Cl OSC(oxidosqualene cyclase)was critical for the negative selection of cucurbitacins during watermelon evolution.This research provides an important resource for metabolomics-assisted breeding in watermelon and for exploring metabolic pathway regulation mechanisms.
基金financially supported by Beijing Academy of Agricultural and Forestry Sciences(KJCX20200204)the National Natural Science Foundation of China(1902034,32122077,and 32172592)+3 种基金the Beijing Scholar Program(YBSP019)the Sci&Tech Innovation of BAAFS(KJCX20200113)the Agriculture Innovation Consortium(BAIC10-2022)the Key Project of Science and Technology of Ningbo(2019B10007)。
文摘ThThe NAC transcription factor NONRIPENING(NOR)is a master regulator of climacteric fruit ripening.Melon(Cucumis melo L.)has climacteric and nonclimacteric fruit ripening varieties and is an ideal model to study fruit ripening.Two natural CmNAC-NOR variants,the climacteric haplotype CmNAC-NOR^(S,N) and the non-climacteric haplotype CmNAC-NOR^(A,S),have effects on fruit ripening;however,their regulatory mechanisms have not been elucidated.Here,we report that a natural mutation in the transcriptional activation domain of CmNAC-NORS,Ncontributes to climacteric melon fruit ripening.CmNAC-NOR knockout in the climacteric-type melon cultivar“BYJH”completely inhibited fruit ripening,while ripening was delayed by 5-8 d in heterozygous cmnac-nor mutant fruits.CmN AC-NOR directly activated carotenoid,ethylene,and abscisic acid biosynthetic genes to promote fruit coloration and ripening.Furthermore,CmNAC-NOR mediated the transcription of the“CmNAC-NOR-CmNAC73-CmCWINV2”module to enhance flesh sweetness.The transcriptional activation activity of the climacteric haplotype CmNAC-NORS,Non these target genes was significantly higher than that of the nonclimacteric haplotype CmNAC-NOR^(A,S).Moreover,CmNAC-NORS,Ncomplementation fully rescued the non-ripening phenotype of the tomato(Solanum lycopersicum)cr-nor mutant,while CmNAC-NOR^(A,S) did not.Our results provide insight into the molecular mechanism of climacteric and non-climacteric fruit ripening in melon.