Watermelon fruit undergoes distinct development stages with dramatic changes during fruit ripening.To date,the molecular mechanics of watermelon ripening remain unclear.Genetic and transcriptome evidences suggested th...Watermelon fruit undergoes distinct development stages with dramatic changes during fruit ripening.To date,the molecular mechanics of watermelon ripening remain unclear.Genetic and transcriptome evidences suggested that the ethylene response factor(ERF)gene ClERF069 may be an important candidate factor affecting watermelon fruit ripening.To dissect the roles of ClERF069 in fruit ripening,structure and phylogenetic analysis were performed using the amplified full-length sequence.Normal-ripening watermelon 97103,non-ripening watermelon PI296341-FR and the RIL population were used to analyze ClERF069 expression dynamics and the correlation with fruit ripening indexs.The results indicated that ClERF069 belongs to ERF family group VI and show high homology(83%identity)to melon ERF069-like protein.ClERF069 expression in watermelon flesh was negatively correlated with fruit lycopene content and sugar content during fruit ripening progress.Further transgenic evidences indicated that overexpression of 35S:ClERF069 in tomato noticeably delayed the ripening process up to 5.2 days.Lycopene,β-carotenoid accumulation patterns were altered and ethylene production patterns in transgenic fruits was significantly delayed during fruit ripening.Taken together,watermelon ethylene response factor ClERF069 was concluded to be a negative regulator of fruit ripening.展开更多
In this study,we used the modified CRISPR/Cas9 system to produce targeted point mutations in cauliflower.Acetolactate synthase(ALS)and Centromere-specific histone H3 variant(CENH3)genes were selected as the base-editi...In this study,we used the modified CRISPR/Cas9 system to produce targeted point mutations in cauliflower.Acetolactate synthase(ALS)and Centromere-specific histone H3 variant(CENH3)genes were selected as the base-editing targets and hypocotyls of cauliflower were used as explants.For ALS gene,a C-to-T conversion in the Pro182 codon(CCT)can alter the encoded amino acid,likely resulting in herbicide resistance,and a C-to-T mutation in the Leu133 codon(CTT)in the CENH3 gene may produce a haploid inducer.Results indicated that the transformation efficiency was 1.8%–4.5%and the mutation efficiencies for the ALS and CENH3 genes were approximately 22%and 87%,respectively.The ALS mutant cauliflower showed strong herbicide resistance,with possible immediate implications for broadleaf weed control in cauliflower fields.展开更多
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
Watermelon(Citrullus lanatus) as non-climacteric fruit is domesticated from the ancestors with inedible fruits. We previously revealed that the abscisic acid(ABA) signaling pathway gene ClSnRK2.3 might infuence waterm...Watermelon(Citrullus lanatus) as non-climacteric fruit is domesticated from the ancestors with inedible fruits. We previously revealed that the abscisic acid(ABA) signaling pathway gene ClSnRK2.3 might infuence watermelon fruit ripening. However,the molecular mechanisms are unclear. Here,we found that the selective variation of ClSnRK2.3 resulted in lower promoter activity and gene expression level in cultivated watermelons than ancestors, which indicated ClSnRK2.3 might be a negative regulator in fruit ripening. Overexpression (OE) of ClSnRK2.3 significantly delayed watermelon fruit ripening and suppressed the accumulation of sucrose, ABA and gibberellin GA4. Furthermore,we determined that the pyrophosphate-dependent phosphofructokinase(ClPFP1) in sugar metabolism pathway and GA biosynthesis enzyme GA20 oxidase(Cl GA20ox) could be phosphorylated by ClSnRK2.3 and thereby resulting in accelerated protein degradation in OE lines and finally led to low levels of sucrose and GA4. Besides that, ClSnRK2.3 phosphorylated homeodomain-leucine zipper protein(ClHAT1) and protected it from degradation to suppress the expression of the ABA biosynthesis gene 9’-cis-epoxycarotenoid dioxygenase 3(Cl NCED3). These results indicated that ClSnRK2.3 negatively regulated watermelon fruit ripening by manipulating the biosynthesis of sucrose, ABA and GA4. Altogether, these findings revealed a novel regulatory mechanism in non-climacteric fruit development and ripening.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
Summary Although it has been reported previously that ethylene plays a critical role in sex determination in cucurbit species, how the andromonoecy that carries both the male and hermaphroditic flowers is determined i...Summary Although it has been reported previously that ethylene plays a critical role in sex determination in cucurbit species, how the andromonoecy that carries both the male and hermaphroditic flowers is determined in watermelon is still unknown. Here we showed that the watermelon gene 1-aminocyclopropane-1-carboxylate syn- thase 4 CCitACS4), expressed specifically in carpel primor- dia, determines the andromonoecy in watermelon. Among four single nucleotide polymorphism (SNPs) and one lnDel identified in the coding region of CitACS4, the C364W mutation located in the conserved box 6 was co- segregated with andromonoecy. Enzymatic analyses showed that the C364W mutation caused a reduced activity in CitACS4. We believe that the reduced CitACS4 activity may hamper the programmed cell death in stamen primordia, leading to the formation of hermaphroditic flowers.展开更多
基金This work was financially supported by the National Key R&D Program of China(Grant No.2018YFD0100703)the Beijing Municipal Science and Technology Project(Grant No.D171100007617001)+4 种基金the Beijing Academy of Agricultural and Forestry Sciences(Grant Nos.QNJJ201733,KJCX20200202)the Ministry of Agriculture and Rural Affairs of China(Grant No.CARS-25)the Beijing Scholar Program(Grant No.BSP026)Beijing Innovation Consortium of Agriculture Research System(Grant No.BAIC10-2020)the Bagui Scholar Program(Grant No.2016A11).
文摘Watermelon fruit undergoes distinct development stages with dramatic changes during fruit ripening.To date,the molecular mechanics of watermelon ripening remain unclear.Genetic and transcriptome evidences suggested that the ethylene response factor(ERF)gene ClERF069 may be an important candidate factor affecting watermelon fruit ripening.To dissect the roles of ClERF069 in fruit ripening,structure and phylogenetic analysis were performed using the amplified full-length sequence.Normal-ripening watermelon 97103,non-ripening watermelon PI296341-FR and the RIL population were used to analyze ClERF069 expression dynamics and the correlation with fruit ripening indexs.The results indicated that ClERF069 belongs to ERF family group VI and show high homology(83%identity)to melon ERF069-like protein.ClERF069 expression in watermelon flesh was negatively correlated with fruit lycopene content and sugar content during fruit ripening progress.Further transgenic evidences indicated that overexpression of 35S:ClERF069 in tomato noticeably delayed the ripening process up to 5.2 days.Lycopene,β-carotenoid accumulation patterns were altered and ethylene production patterns in transgenic fruits was significantly delayed during fruit ripening.Taken together,watermelon ethylene response factor ClERF069 was concluded to be a negative regulator of fruit ripening.
基金partly funded by the project of technology innovation ability from Beijing Academy of Agriculture and Forestry Sciences (Grant Nos. KJCX20200401, KJCX20200205 and KJCX20200113)the Natural Science Foundation of China (Grant No. 31972401)
文摘In this study,we used the modified CRISPR/Cas9 system to produce targeted point mutations in cauliflower.Acetolactate synthase(ALS)and Centromere-specific histone H3 variant(CENH3)genes were selected as the base-editing targets and hypocotyls of cauliflower were used as explants.For ALS gene,a C-to-T conversion in the Pro182 codon(CCT)can alter the encoded amino acid,likely resulting in herbicide resistance,and a C-to-T mutation in the Leu133 codon(CTT)in the CENH3 gene may produce a haploid inducer.Results indicated that the transformation efficiency was 1.8%–4.5%and the mutation efficiencies for the ALS and CENH3 genes were approximately 22%and 87%,respectively.The ALS mutant cauliflower showed strong herbicide resistance,with possible immediate implications for broadleaf weed control in cauliflower fields.
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financially supported by the National Natural Science Foundation of China (31930096, 31902034, 32172592, 32122077, and 32072601)Foundation of Beijing Academy of Agricultural and Forestry Sciences (QNJJ202032 and QNJJ202206)+4 种基金the Ministry of Agriculture and Rural Affairs of China (CARS-25)the Beijing Scholar Program (YBSP019)Foundation of Xinjiang production and construction corps (2022AB015)The Agriculture Innovation Consortium (BAIC04-2023)The Scientist Training Program of BAAFS (JKZX202211)。
文摘Watermelon(Citrullus lanatus) as non-climacteric fruit is domesticated from the ancestors with inedible fruits. We previously revealed that the abscisic acid(ABA) signaling pathway gene ClSnRK2.3 might infuence watermelon fruit ripening. However,the molecular mechanisms are unclear. Here,we found that the selective variation of ClSnRK2.3 resulted in lower promoter activity and gene expression level in cultivated watermelons than ancestors, which indicated ClSnRK2.3 might be a negative regulator in fruit ripening. Overexpression (OE) of ClSnRK2.3 significantly delayed watermelon fruit ripening and suppressed the accumulation of sucrose, ABA and gibberellin GA4. Furthermore,we determined that the pyrophosphate-dependent phosphofructokinase(ClPFP1) in sugar metabolism pathway and GA biosynthesis enzyme GA20 oxidase(Cl GA20ox) could be phosphorylated by ClSnRK2.3 and thereby resulting in accelerated protein degradation in OE lines and finally led to low levels of sucrose and GA4. Besides that, ClSnRK2.3 phosphorylated homeodomain-leucine zipper protein(ClHAT1) and protected it from degradation to suppress the expression of the ABA biosynthesis gene 9’-cis-epoxycarotenoid dioxygenase 3(Cl NCED3). These results indicated that ClSnRK2.3 negatively regulated watermelon fruit ripening by manipulating the biosynthesis of sucrose, ABA and GA4. Altogether, these findings revealed a novel regulatory mechanism in non-climacteric fruit development and ripening.
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.
基金supported by grants from National Natural Science Foundation (31361140355, 31401893, 31272184)National Key Development Plan Pilot Projects (Functional genomics research and application of wheat and other crops)+3 种基金Beijing Scholar Program and Beijing Excellent Talents Program (2014000021223TD03)Beijing Municipal Science & Technology Commission (6141001, 6144023, WRDMC01)Ministry of Science and Technology of China (2015BAD02B0202, 2014BAD01B08)Ministry of Agriculture of China (CARS-26)
文摘Summary Although it has been reported previously that ethylene plays a critical role in sex determination in cucurbit species, how the andromonoecy that carries both the male and hermaphroditic flowers is determined in watermelon is still unknown. Here we showed that the watermelon gene 1-aminocyclopropane-1-carboxylate syn- thase 4 CCitACS4), expressed specifically in carpel primor- dia, determines the andromonoecy in watermelon. Among four single nucleotide polymorphism (SNPs) and one lnDel identified in the coding region of CitACS4, the C364W mutation located in the conserved box 6 was co- segregated with andromonoecy. Enzymatic analyses showed that the C364W mutation caused a reduced activity in CitACS4. We believe that the reduced CitACS4 activity may hamper the programmed cell death in stamen primordia, leading to the formation of hermaphroditic flowers.