Huanglongbing(HLB)is the most devastating disease for citrus worldwide.Candidatus Liberibacter asiaticus(C Las),vectored by Asian citrus psyllid(ACP,Diaphorina citri Kuwayama),is the most common pathogen causing the d...Huanglongbing(HLB)is the most devastating disease for citrus worldwide.Candidatus Liberibacter asiaticus(C Las),vectored by Asian citrus psyllid(ACP,Diaphorina citri Kuwayama),is the most common pathogen causing the disease.Commercial citrus varieties are highly susceptible to HLB,whereas trifoliate orange(Poncirus trifoliata)is considered highly tolerant to HLB.An F1 segregating population and their parent trifoliate orange and sweet orange,which had been exposed to intense HLB pressure for three years,was evaluated for disease symptoms,ACP colonization,C Las titer and tree vigor repeatedly for two to three years.Trifoliate orange and sweet orange showed significant differences for most of the phenotypic traits,and the F1 population exhibited a large variation.A high-density SNP-based genetic map with 1402 markers was constructed for trifoliate orange,which exhibited high synteny and high coverage of its reference genome.A total of 26 quantitative trait locus(QTLs)were identified in four linkage groups LG-t6,LG-t7,LG-t8 and LG-t9,of which four QTL clusters exhibit a clear co-localization of QTLs associated with different traits.Through genome-wide analysis of gene expression in response to C Las infection in‘Flying Dragon’and‘Larger-Flower DPI-50-7’trifoliate orange,85 differentially expressed genes were found located within the QTL clusters.Among them,seven genes were classified as defense or immunity protein which exhibited the highest transcriptional change after C Las infection.Our results indicate a quantitative genetic nature of HLB tolerance and identified candidate genes that should be valuable for searching for genetic solutions to HLB through breeding or genetic engineering.展开更多
Fruits and vegetables are an essential part of a healthy diet, providing humans with vitamins, phytonutrients, and minerals. They are significantly vulnerable, however, to post-harvest diseases caused by numerous fung...Fruits and vegetables are an essential part of a healthy diet, providing humans with vitamins, phytonutrients, and minerals. They are significantly vulnerable, however, to post-harvest diseases caused by numerous fungal and bacterial pathogens. These pathogens can cause significant quantitative and qualitative losses from harvest to consumption during the handling and storage processes. Chemical fungicides are commonly used but are likely to leave residues on the produce, rendering short shelf-life produce, such as berries, unsuitable for human consumption. Identifying eco-friendly methods to control post-harvest disease is, therefore, of utmost importance. The presence of antifungal constituents in the roots of Poncirus trifoliata extracts was detected by thin layer chromatography-based bioautography. The active constituents were isolated and identified by bioautography assay-guided fractionation using flash chromatography followed by spectroscopic techniques. In this study, xanthoxyletin, demethylsuberosin, dentatin, nordentatin, ponfolin, and clausarin were isolated from the root extracts. The antifungal activity of these compounds was moderate to weak compared to the commercial fungicide captan. This study reports the isolation and identification of natural compounds from Poncirus trifoliata that exhibited antifungal activity against Colletotrichum fragariae and Botrytis cinerea, two major post-harvest pathogens.展开更多
Huanglongbing(HLB)is the most destructive bacterial disease of citrus worldwide.While most citrus varieties are susceptible to HLB,Poncirus trifoliata,a close relative of Citrus,and some of its hybrids with Citrus are...Huanglongbing(HLB)is the most destructive bacterial disease of citrus worldwide.While most citrus varieties are susceptible to HLB,Poncirus trifoliata,a close relative of Citrus,and some of its hybrids with Citrus are tolerant to HLB.No specific HLB tolerance genes have been identified in P.trifoliata but recent studies have shown that constitutive disease resistance(CDR)genes were expressed at much higher levels in HLB-tolerant Poncirus hybrids and the expression of CDR genes was modulated by Candidatus Liberibacter asiaticus(CLas),the pathogen of HLB.The current study was undertaken to mine and characterize the CDR gene family in Citrus and Poncirus and to understand its association with HLB tolerance in Poncirus.We identified 17 CDR genes in two citrus genomes,deduced their structures,and investigated their phylogenetic relationships.We revealed that the expansion of the CDR family in Citrus seems to be due to segmental and tandem duplication events.Through genome resequencing and transcriptome sequencing,we identified eight CDR genes in the Poncirus genome(PtCDR1-PtCDR8).The number of SNPs was the highest in PtCDR2 and the lowest in PtCDR7.Most of the deletion and insertion events were observed in the UTR regions of Citrus and Poncirus CDR genes.PtCDR2 and PtCDR8 were in abundance in the leaf transcriptomes of two HLB-tolerant Poncirus genotypes and were also upregulated in HLB-tolerant,Poncirus hybrids as revealed by real-time PCR analysis.These two CDR genes seem to be good candidate genes for future studies of their role in citrus-CLas interactions.展开更多
Citrus canker, caused by Xanthomonas axonopo-dis pv. citri ('Xac'), is an important quarantine disease in citrus crops. Arbuscular mycorrhizal fungi (AMF) form symbiotic interactions with host plants and furth...Citrus canker, caused by Xanthomonas axonopo-dis pv. citri ('Xac'), is an important quarantine disease in citrus crops. Arbuscular mycorrhizal fungi (AMF) form symbiotic interactions with host plants and further affect their disease resistance, possibly by modulating the activity of salicylic acid (SA), a key phytohormone in disease resistance. Common mycorrhizal networks (CMNs) can interconnect plants, but it is not yet clear whether CMNs promote resistance to citrus canker and, if so, whether SA signaling is involved in this process. To test this possibility, we used a two-chambered rootbox to establish CMNs between trifoliate orange (Poncirus trifoliata) seedlings in chambers inoculated (treated) or not (neighboring) with the AMF, Paraglomus occultum. A subset of the AMF-inoculated seedlings were also inoculated with Xac (+AMF+Xac). At 2 d post-inoculation (dpi), compared with the +AMF-Xac treatment, neighboring seedlings in +AMF+Xac treatment had lower expression levels of the SA biosynthetic genes, PtPAL, PtEPS1, and PtPBS3, but higher SA levels, which attributed to the upregulation of PtPAL and PtPBS3 in treated seedlings and the transfer of SA, via CMNs, to the neighboring seedlings. At 4 dpi, the pathogenesis-related (PR) protein genes, PtPR1, PtPR4, and PtPR5, and the transcriptional regulatory factor gene, PtNPR1, were activated in neighboring seedlings of+AMF+Xac treatment. At 9 dpi, root phenylalanine ammo-nia-lyase activity and total soluble phenol and lignin concentrations increased in neighboring seedlings of+AMF+Xac treatment, likely due to the linkage and signal transfer, via CMNs. These findings support the hypothesis that CMNs transfer the SA signal from infected to neighboring healthy seedlings, to activate defense responses and affording protection to neighboring plants against citrus canker infection.展开更多
Polyploids have elevated stress tolerance,but the underlying mechanisms remain largely elusive.In this study,we showed that naturally occurring tetraploid plants of trifoliate orange(Poncirus trifoliata(L.) Raf.) exhi...Polyploids have elevated stress tolerance,but the underlying mechanisms remain largely elusive.In this study,we showed that naturally occurring tetraploid plants of trifoliate orange(Poncirus trifoliata(L.) Raf.) exhibited enhanced cold tolerance relative to their diploid progenitors.Transcriptome analysis revealed that whole-genome duplication was associated with higher expression levels of a range of well-characterized cold stress-responsive genes.Global DNA methylation profiling demonstrated that the tetraploids underwent more extensive DNA demethylation in comparison with the diploids under cold stress.CHH methylation in the promoters was associated with up-regulation of related genes,whereas CG,CHG,and CHH methylation in the 3’-regions was relevant to gene down-regulation.Of note,genes involved in unsaturated fatty acids(UFAs) and jasmonate(JA)biosynthesis in the tetraploids displayed different CHH methylation in the gene flanking regions and were prominently up-regulated,consistent with greater accumulation of UFAs and JA when exposed to the cold stress.Collectively,our findings explored the difference in cold stress response between diploids and tetraploids at both transcriptional and epigenetic levels,and gained new insight into the molecular mechanisms underlying enhanced cold tolerance of the tetraploid.These results contribute to uncovering a novel regulatory role of DNA methylation in better cold tolerance of polyploids.展开更多
Late embryogenesis abundant(LEA)genes encode highly hydrophilic proteins that are essential in abiotic stress responses.However,most LEA genes in higher plants have not yet been investigated.This study identified an L...Late embryogenesis abundant(LEA)genes encode highly hydrophilic proteins that are essential in abiotic stress responses.However,most LEA genes in higher plants have not yet been investigated.This study identified an LEA family gene(PtrLEA7)from Poncirus trifoliata and studied its function in drought tolerance.The full-length coding sequence of PtrLEA7 was 420 bp encoding a protein of 139 amino acids.Phylogenetic analysis shows that PtrLEA7 protein belongs to the LEA_4 subfamily.Expression profiling by qPCR found that PtrLEA7 was strongly induced by dehydration,cold and ABA treatments,and slightly induced by salt stress.Subcellular localization reveals that PtrLEA7 protein was located in both cytoplasm and nucleus.To investigate its function,transgenic plants of both tobacco and Poncirus trifoliata overexpressing PtrLEA7 were obtained.Stress tolerance assays show that overexpression lines had enhanced dehydration and drought tolerance compared with wild type plants,indicating that PtrLEA7 positively regulates drought tolerance.In addition,transgenic plants had much higher expression levels of three antioxidant enzyme genes(CAT,SOD and POD)and significantly increased catalase enzyme activity,accompanied by reduced reactive oxygen species accumulation in comparison with wild type plants.Collectively,this study demonstrates that PtrLEA7 can confer enhanced drought tolerance partially via enhancing antioxidant capacity.展开更多
Based on the former constructed ＇Tyrl＇ locus genetic map in family 9145, from LB6-2 [Clementine mandarin （C. reticulata） × Hamlin orange （C. sinensis）] × Swingle citrumelo （C. paradise × P. trifo...Based on the former constructed ＇Tyrl＇ locus genetic map in family 9145, from LB6-2 [Clementine mandarin （C. reticulata） × Hamlin orange （C. sinensis）] × Swingle citrumelo （C. paradise × P. trifoliata）, 9 markers were chosen for application in evaluating their effectiveness in marker-assisted selection （MAS） for citrus rootstock breeding program from many F1 progeny of Poncirus trifoliata. As the mapping revealed that these markers were estimated within a range of 12.1 cM in the linkage group, and among them, SCO07 co-segregated with ＂Tyrl＇, and 7A4R as the closest to ＇Tyrl＇ with a distance of 1.5 cM, these markers were basically fitful to go MAS screening. The results of screening P. trifoliata F1 progeny indicated that all the markers were inherited in codominant fashion and most of them were heterozygous on PT （Pomery of P. trifoliata）., marker 4L17R/CfoI and 7A4（1407）/BfaI were proved to be consistently reliable for accurate scoring of genotypes and the revealed polymorphism was basically coincided with the citrus nematode resistant phenotype within tested populations. The polymorphic genotype with marker 4L17R/Cfol was found completely matched up with the phenotype of individuals that conferred high resistance to citrus nematode when the USDA hybrid rootstocks were screened. Utilization of these markers, especially the highly specific 4L17R/Cfol and 7A4（1407）/Bfal, should result in great benefit to world citrus industry for early selection in rootstock-breeding program.展开更多
Novel Poncirus trifoliata simple sequence repeat(SSR) markers were developed to evaluate their utility for genetic diversity and breeding studies of P. trifoliata and related species. A total of 108 primer pairs were ...Novel Poncirus trifoliata simple sequence repeat(SSR) markers were developed to evaluate their utility for genetic diversity and breeding studies of P. trifoliata and related species. A total of 108 primer pairs were characterized by PCR amplification experiments. Among these, 61 were polymorphic and transferable to other citrus species. The number of alleles per locus ranged from 2 to 6, with an average of 2.37 alleles per locus. The expected heterozygosity and observed heterozygosity ranged from 0 to 0.83 and 0 to 1.00, respectively.These novel polymorphic SSR markers will be useful for citrus cultivar identification and evaluation as well as breeding studies.展开更多
Forsythia fructus has been shown to have antioxidative, anti-inflammatory, antibacterial, anti-aging and whitening effects. Hoechunyangkyeok-san (Forsythia viridissima-prescription) is a traditional herbal medicine, w...Forsythia fructus has been shown to have antioxidative, anti-inflammatory, antibacterial, anti-aging and whitening effects. Hoechunyangkyeok-san (Forsythia viridissima-prescription) is a traditional herbal medicine, which has been clinically used for treating febrile and inflammatory disorders. This work was carried out to investigate the skin whitening effects of Forsythia viridissima-prescription extract (a hydrolyzed extract of Hoechunyangkyeok-san: SID White HYC) on skin. The effects of SID White HYC were assessed the melanin contents in B161 melanoma cells and the pigmented equivalent with HMB45 and Fontana Masson staining in 3D skin model. Then, we examined the expression of major pigment enzymes regulating melanin synthesis and melanosome transport related proteins in B16F1 cells. SID White HYC significantly inhibited the melanin synthesis (56.7% and 30.6% inhibition at 100 μg/mL, intracellular and secreted, respectively) in B16F1 cells and 3D skin model. In addition, western blotting analysis showed that SID White HYC reduced the expression of melanin synthesis and melanosome transport related proteins in B16F1 cells. In clinical trials, the cream containing 0.05% SID White HYC showed skin depigmentation effect without any irritation. These results suggest that SID White HYC may be useful inhibition of melanogenesis and melanosome transport. Therefore, SID White HYC may have potential as a skin-whitening ingredient in cosmetics.展开更多
It has been hypothesized that under iron stress high ferric chelate reductase (FCR) activity in the absorptive root of plants tolerant to iron_deficiency will be induced and result in subsequent Fe 2+ transport a...It has been hypothesized that under iron stress high ferric chelate reductase (FCR) activity in the absorptive root of plants tolerant to iron_deficiency will be induced and result in subsequent Fe 2+ transport across the plasmalemma. The activity of FCR and expression of FCR gene (FRO2) in Citrus junos Sieb. ex Tanaka tolerant to iron_deficiency and Poncirus trifoliata (L.) Raf. susceptible to iron_deficiency were determined to elucidate the physiological difference which causes the different tolerance of the two citrus rootstocks to iron stress. The activity of FCR was detectable in excised roots and was stimulated about 20_times in C. junos and only about 3_times in P. trifoliata under iron deficiency for four weeks. The FRO2 of Arabidopsis was used as a probe, the tissue print technique was used to ascertain the expression of the FCR gene in C. junos and P. trifoliata under iron stress. High_level transcripts were observed in the absorptive root, young green stem as well as new leaf of C. junos under iron stress for two weeks, and the transcripts were accumulated only slightly in P. trifoliata at the same time. The results showed that the obvious increase of FCR activity was an important reason for the tolerance of C. junos to iron_deficiency, and the regulation of FCR activity seemed to be at the transcriptional level, and the expression of FRO2 occurred in the root, stem and leaf.展开更多
The local auxin distribution characteristics in the roots,stems,and leaves of stably transformed plantlets of trifoliate orange(Poncirus trifoliata)with auxin reporter system DR5::GUS-YFP were elucidated in this resea...The local auxin distribution characteristics in the roots,stems,and leaves of stably transformed plantlets of trifoliate orange(Poncirus trifoliata)with auxin reporter system DR5::GUS-YFP were elucidated in this research.The auxin response maxima could be observed in the apex of the root tip,primary phloem of the tender stem,and the margin of the young leaves according to the activity of theβ-glucuronidase(GUS)reporter gene triggered by the auxin responsive DR5 promoter.Auxin responses in the apex of the root tips increased when treated with synthetic auxin 1-naphthylacetic acid(NAA),but decreased when treated with the auxin polar transportation inhibitor 2,3,5-triiodobenzoic acid(TIBA).These results indicated that the DR5 reporter system worked in P.trifoliata for auxin distribution and response observation.Trifoliate orange is highly susceptible to citrus canker disease.Auxin accumulation was observed visually in the invasion sites of the detached leaves inoculated with Xanthomonas axonopodis pv.citri(Xac)by GUS staining;the upregulated expression of the YFP,GH3.1,GH3.9,and SAUR genes assessed by quantitative real-time PCR(qRT-PCR)also identified auxin accumulation in the inoculated tissues following Xac infection.Overall,these findings indicated that the plantlets of P.trifoliata engineered with the auxin reporter gene provided a promising system for studying auxin responses during Xac infection.展开更多
In the framework of searching for new pectin sources to partially compensate for domestic and regional demands, the peel (albedo) of the “non-comestible” fruit of Poncirus trifoliata was investigated using a relativ...In the framework of searching for new pectin sources to partially compensate for domestic and regional demands, the peel (albedo) of the “non-comestible” fruit of Poncirus trifoliata was investigated using a relatively simple experimental design for optimization, in which only the variable was the extraction pH (1.0, 1.5, and 2.0) on the basis of our previous studies on diverse pectin sources. The results showed that the yield of pectin (7.4%-19.8%) was strongly influenced by the extraction pH when the other parameters, namely the solid to liquid extractant (S/L) ratio, temperature (T °C), and time (t) were fixed to 1:25 (w/v), 75°C, and 90 min, respectively. Likewise, the galacturonic acid content (GalA: 61.4%-79.2%), total neutral sugar content (TNS: 9.1%-22.5%), degree of branching (3.5%-13.9%), homogalacturonan (HG) to rhamnogalacturonan-I (RG-I) ratio (2.2-5.6), degree of methylesterification (DM: 54-77), viscosity average molecular weight (Mν: 57-82), and gelling capacity (GC: 124-158) were all affected by the extraction pH. The optimum pH for producing pectin with good yield, quality characteristics (GalA > 65%, DM > 60, Mν > 80 kDa), and gelling capacity (GC > 150), from the peel of P. trifoliata fruit, was found to be pH 1.5.展开更多
基金supported by grants from the Citrus Research and Development Foundation,USA(Grant No.CRDF#15-010)the New Varieties Development and Management Corporation(NVDMC),on behalf of the Florida citrus industry,USA,the Fundamental Research Funds for the Central Universities,China(Grant No.2022CDJXY-004)from the USDA-NIFA-SCRI,USA(Grant No.2015-70016-2302).
文摘Huanglongbing(HLB)is the most devastating disease for citrus worldwide.Candidatus Liberibacter asiaticus(C Las),vectored by Asian citrus psyllid(ACP,Diaphorina citri Kuwayama),is the most common pathogen causing the disease.Commercial citrus varieties are highly susceptible to HLB,whereas trifoliate orange(Poncirus trifoliata)is considered highly tolerant to HLB.An F1 segregating population and their parent trifoliate orange and sweet orange,which had been exposed to intense HLB pressure for three years,was evaluated for disease symptoms,ACP colonization,C Las titer and tree vigor repeatedly for two to three years.Trifoliate orange and sweet orange showed significant differences for most of the phenotypic traits,and the F1 population exhibited a large variation.A high-density SNP-based genetic map with 1402 markers was constructed for trifoliate orange,which exhibited high synteny and high coverage of its reference genome.A total of 26 quantitative trait locus(QTLs)were identified in four linkage groups LG-t6,LG-t7,LG-t8 and LG-t9,of which four QTL clusters exhibit a clear co-localization of QTLs associated with different traits.Through genome-wide analysis of gene expression in response to C Las infection in‘Flying Dragon’and‘Larger-Flower DPI-50-7’trifoliate orange,85 differentially expressed genes were found located within the QTL clusters.Among them,seven genes were classified as defense or immunity protein which exhibited the highest transcriptional change after C Las infection.Our results indicate a quantitative genetic nature of HLB tolerance and identified candidate genes that should be valuable for searching for genetic solutions to HLB through breeding or genetic engineering.
文摘Fruits and vegetables are an essential part of a healthy diet, providing humans with vitamins, phytonutrients, and minerals. They are significantly vulnerable, however, to post-harvest diseases caused by numerous fungal and bacterial pathogens. These pathogens can cause significant quantitative and qualitative losses from harvest to consumption during the handling and storage processes. Chemical fungicides are commonly used but are likely to leave residues on the produce, rendering short shelf-life produce, such as berries, unsuitable for human consumption. Identifying eco-friendly methods to control post-harvest disease is, therefore, of utmost importance. The presence of antifungal constituents in the roots of Poncirus trifoliata extracts was detected by thin layer chromatography-based bioautography. The active constituents were isolated and identified by bioautography assay-guided fractionation using flash chromatography followed by spectroscopic techniques. In this study, xanthoxyletin, demethylsuberosin, dentatin, nordentatin, ponfolin, and clausarin were isolated from the root extracts. The antifungal activity of these compounds was moderate to weak compared to the commercial fungicide captan. This study reports the isolation and identification of natural compounds from Poncirus trifoliata that exhibited antifungal activity against Colletotrichum fragariae and Botrytis cinerea, two major post-harvest pathogens.
基金ZD acknowledges financial support of this study from the Citrus Research and Development Foundation,Inc.(CDRF)(Project#108766 and#105077)from the USDA-NIFA Citrus Disease Research and Extension(CDRE)Program(Grant No.2015-70016-23027).
文摘Huanglongbing(HLB)is the most destructive bacterial disease of citrus worldwide.While most citrus varieties are susceptible to HLB,Poncirus trifoliata,a close relative of Citrus,and some of its hybrids with Citrus are tolerant to HLB.No specific HLB tolerance genes have been identified in P.trifoliata but recent studies have shown that constitutive disease resistance(CDR)genes were expressed at much higher levels in HLB-tolerant Poncirus hybrids and the expression of CDR genes was modulated by Candidatus Liberibacter asiaticus(CLas),the pathogen of HLB.The current study was undertaken to mine and characterize the CDR gene family in Citrus and Poncirus and to understand its association with HLB tolerance in Poncirus.We identified 17 CDR genes in two citrus genomes,deduced their structures,and investigated their phylogenetic relationships.We revealed that the expansion of the CDR family in Citrus seems to be due to segmental and tandem duplication events.Through genome resequencing and transcriptome sequencing,we identified eight CDR genes in the Poncirus genome(PtCDR1-PtCDR8).The number of SNPs was the highest in PtCDR2 and the lowest in PtCDR7.Most of the deletion and insertion events were observed in the UTR regions of Citrus and Poncirus CDR genes.PtCDR2 and PtCDR8 were in abundance in the leaf transcriptomes of two HLB-tolerant Poncirus genotypes and were also upregulated in HLB-tolerant,Poncirus hybrids as revealed by real-time PCR analysis.These two CDR genes seem to be good candidate genes for future studies of their role in citrus-CLas interactions.
基金supported by the Plan in Scientific and Technological Innovation Team of Outstanding Young Scientists, Hubei Provincial Department of Education (T201604)
文摘Citrus canker, caused by Xanthomonas axonopo-dis pv. citri ('Xac'), is an important quarantine disease in citrus crops. Arbuscular mycorrhizal fungi (AMF) form symbiotic interactions with host plants and further affect their disease resistance, possibly by modulating the activity of salicylic acid (SA), a key phytohormone in disease resistance. Common mycorrhizal networks (CMNs) can interconnect plants, but it is not yet clear whether CMNs promote resistance to citrus canker and, if so, whether SA signaling is involved in this process. To test this possibility, we used a two-chambered rootbox to establish CMNs between trifoliate orange (Poncirus trifoliata) seedlings in chambers inoculated (treated) or not (neighboring) with the AMF, Paraglomus occultum. A subset of the AMF-inoculated seedlings were also inoculated with Xac (+AMF+Xac). At 2 d post-inoculation (dpi), compared with the +AMF-Xac treatment, neighboring seedlings in +AMF+Xac treatment had lower expression levels of the SA biosynthetic genes, PtPAL, PtEPS1, and PtPBS3, but higher SA levels, which attributed to the upregulation of PtPAL and PtPBS3 in treated seedlings and the transfer of SA, via CMNs, to the neighboring seedlings. At 4 dpi, the pathogenesis-related (PR) protein genes, PtPR1, PtPR4, and PtPR5, and the transcriptional regulatory factor gene, PtNPR1, were activated in neighboring seedlings of+AMF+Xac treatment. At 9 dpi, root phenylalanine ammo-nia-lyase activity and total soluble phenol and lignin concentrations increased in neighboring seedlings of+AMF+Xac treatment, likely due to the linkage and signal transfer, via CMNs. These findings support the hypothesis that CMNs transfer the SA signal from infected to neighboring healthy seedlings, to activate defense responses and affording protection to neighboring plants against citrus canker infection.
基金National Key Research and Development Program of China(2018YFD1000302)the National Natural Science Foundation of China(31972377)。
文摘Polyploids have elevated stress tolerance,but the underlying mechanisms remain largely elusive.In this study,we showed that naturally occurring tetraploid plants of trifoliate orange(Poncirus trifoliata(L.) Raf.) exhibited enhanced cold tolerance relative to their diploid progenitors.Transcriptome analysis revealed that whole-genome duplication was associated with higher expression levels of a range of well-characterized cold stress-responsive genes.Global DNA methylation profiling demonstrated that the tetraploids underwent more extensive DNA demethylation in comparison with the diploids under cold stress.CHH methylation in the promoters was associated with up-regulation of related genes,whereas CG,CHG,and CHH methylation in the 3’-regions was relevant to gene down-regulation.Of note,genes involved in unsaturated fatty acids(UFAs) and jasmonate(JA)biosynthesis in the tetraploids displayed different CHH methylation in the gene flanking regions and were prominently up-regulated,consistent with greater accumulation of UFAs and JA when exposed to the cold stress.Collectively,our findings explored the difference in cold stress response between diploids and tetraploids at both transcriptional and epigenetic levels,and gained new insight into the molecular mechanisms underlying enhanced cold tolerance of the tetraploid.These results contribute to uncovering a novel regulatory role of DNA methylation in better cold tolerance of polyploids.
基金the National Key Research and Development Program of China(2018YFD1000300)the National Natural Science Foundation of China(31972377)+1 种基金Hubei Provincial Natural Science Foundation for Innovative Group(2017CFA018)Leading Talents Fund in Science and Technology Innovation in Henan Province(194200510007).
文摘Late embryogenesis abundant(LEA)genes encode highly hydrophilic proteins that are essential in abiotic stress responses.However,most LEA genes in higher plants have not yet been investigated.This study identified an LEA family gene(PtrLEA7)from Poncirus trifoliata and studied its function in drought tolerance.The full-length coding sequence of PtrLEA7 was 420 bp encoding a protein of 139 amino acids.Phylogenetic analysis shows that PtrLEA7 protein belongs to the LEA_4 subfamily.Expression profiling by qPCR found that PtrLEA7 was strongly induced by dehydration,cold and ABA treatments,and slightly induced by salt stress.Subcellular localization reveals that PtrLEA7 protein was located in both cytoplasm and nucleus.To investigate its function,transgenic plants of both tobacco and Poncirus trifoliata overexpressing PtrLEA7 were obtained.Stress tolerance assays show that overexpression lines had enhanced dehydration and drought tolerance compared with wild type plants,indicating that PtrLEA7 positively regulates drought tolerance.In addition,transgenic plants had much higher expression levels of three antioxidant enzyme genes(CAT,SOD and POD)and significantly increased catalase enzyme activity,accompanied by reduced reactive oxygen species accumulation in comparison with wild type plants.Collectively,this study demonstrates that PtrLEA7 can confer enhanced drought tolerance partially via enhancing antioxidant capacity.
基金supported in part by grants from the USDA NRICGP (9201765, 9600748)USDA/National Citrus Research Council (98012205)the Florida Citrus Production Research Advisory Council, USA(942-27)
文摘Based on the former constructed ＇Tyrl＇ locus genetic map in family 9145, from LB6-2 [Clementine mandarin （C. reticulata） × Hamlin orange （C. sinensis）] × Swingle citrumelo （C. paradise × P. trifoliata）, 9 markers were chosen for application in evaluating their effectiveness in marker-assisted selection （MAS） for citrus rootstock breeding program from many F1 progeny of Poncirus trifoliata. As the mapping revealed that these markers were estimated within a range of 12.1 cM in the linkage group, and among them, SCO07 co-segregated with ＂Tyrl＇, and 7A4R as the closest to ＇Tyrl＇ with a distance of 1.5 cM, these markers were basically fitful to go MAS screening. The results of screening P. trifoliata F1 progeny indicated that all the markers were inherited in codominant fashion and most of them were heterozygous on PT （Pomery of P. trifoliata）., marker 4L17R/CfoI and 7A4（1407）/BfaI were proved to be consistently reliable for accurate scoring of genotypes and the revealed polymorphism was basically coincided with the citrus nematode resistant phenotype within tested populations. The polymorphic genotype with marker 4L17R/Cfol was found completely matched up with the phenotype of individuals that conferred high resistance to citrus nematode when the USDA hybrid rootstocks were screened. Utilization of these markers, especially the highly specific 4L17R/Cfol and 7A4（1407）/Bfal, should result in great benefit to world citrus industry for early selection in rootstock-breeding program.
基金supported by the Ministry of Science and Technology of China(2011CB100601)the National Natural Science Foundation of China(31330066)
文摘Novel Poncirus trifoliata simple sequence repeat(SSR) markers were developed to evaluate their utility for genetic diversity and breeding studies of P. trifoliata and related species. A total of 108 primer pairs were characterized by PCR amplification experiments. Among these, 61 were polymorphic and transferable to other citrus species. The number of alleles per locus ranged from 2 to 6, with an average of 2.37 alleles per locus. The expected heterozygosity and observed heterozygosity ranged from 0 to 0.83 and 0 to 1.00, respectively.These novel polymorphic SSR markers will be useful for citrus cultivar identification and evaluation as well as breeding studies.
文摘Forsythia fructus has been shown to have antioxidative, anti-inflammatory, antibacterial, anti-aging and whitening effects. Hoechunyangkyeok-san (Forsythia viridissima-prescription) is a traditional herbal medicine, which has been clinically used for treating febrile and inflammatory disorders. This work was carried out to investigate the skin whitening effects of Forsythia viridissima-prescription extract (a hydrolyzed extract of Hoechunyangkyeok-san: SID White HYC) on skin. The effects of SID White HYC were assessed the melanin contents in B161 melanoma cells and the pigmented equivalent with HMB45 and Fontana Masson staining in 3D skin model. Then, we examined the expression of major pigment enzymes regulating melanin synthesis and melanosome transport related proteins in B16F1 cells. SID White HYC significantly inhibited the melanin synthesis (56.7% and 30.6% inhibition at 100 μg/mL, intracellular and secreted, respectively) in B16F1 cells and 3D skin model. In addition, western blotting analysis showed that SID White HYC reduced the expression of melanin synthesis and melanosome transport related proteins in B16F1 cells. In clinical trials, the cream containing 0.05% SID White HYC showed skin depigmentation effect without any irritation. These results suggest that SID White HYC may be useful inhibition of melanogenesis and melanosome transport. Therefore, SID White HYC may have potential as a skin-whitening ingredient in cosmetics.
文摘It has been hypothesized that under iron stress high ferric chelate reductase (FCR) activity in the absorptive root of plants tolerant to iron_deficiency will be induced and result in subsequent Fe 2+ transport across the plasmalemma. The activity of FCR and expression of FCR gene (FRO2) in Citrus junos Sieb. ex Tanaka tolerant to iron_deficiency and Poncirus trifoliata (L.) Raf. susceptible to iron_deficiency were determined to elucidate the physiological difference which causes the different tolerance of the two citrus rootstocks to iron stress. The activity of FCR was detectable in excised roots and was stimulated about 20_times in C. junos and only about 3_times in P. trifoliata under iron deficiency for four weeks. The FRO2 of Arabidopsis was used as a probe, the tissue print technique was used to ascertain the expression of the FCR gene in C. junos and P. trifoliata under iron stress. High_level transcripts were observed in the absorptive root, young green stem as well as new leaf of C. junos under iron stress for two weeks, and the transcripts were accumulated only slightly in P. trifoliata at the same time. The results showed that the obvious increase of FCR activity was an important reason for the tolerance of C. junos to iron_deficiency, and the regulation of FCR activity seemed to be at the transcriptional level, and the expression of FRO2 occurred in the root, stem and leaf.
基金the National Natural Science Foundation of China(Grant No.31660564)the science and technology project of Jiangxi province(Grant Nos.20161BBF60063,151008).
文摘The local auxin distribution characteristics in the roots,stems,and leaves of stably transformed plantlets of trifoliate orange(Poncirus trifoliata)with auxin reporter system DR5::GUS-YFP were elucidated in this research.The auxin response maxima could be observed in the apex of the root tip,primary phloem of the tender stem,and the margin of the young leaves according to the activity of theβ-glucuronidase(GUS)reporter gene triggered by the auxin responsive DR5 promoter.Auxin responses in the apex of the root tips increased when treated with synthetic auxin 1-naphthylacetic acid(NAA),but decreased when treated with the auxin polar transportation inhibitor 2,3,5-triiodobenzoic acid(TIBA).These results indicated that the DR5 reporter system worked in P.trifoliata for auxin distribution and response observation.Trifoliate orange is highly susceptible to citrus canker disease.Auxin accumulation was observed visually in the invasion sites of the detached leaves inoculated with Xanthomonas axonopodis pv.citri(Xac)by GUS staining;the upregulated expression of the YFP,GH3.1,GH3.9,and SAUR genes assessed by quantitative real-time PCR(qRT-PCR)also identified auxin accumulation in the inoculated tissues following Xac infection.Overall,these findings indicated that the plantlets of P.trifoliata engineered with the auxin reporter gene provided a promising system for studying auxin responses during Xac infection.
文摘In the framework of searching for new pectin sources to partially compensate for domestic and regional demands, the peel (albedo) of the “non-comestible” fruit of Poncirus trifoliata was investigated using a relatively simple experimental design for optimization, in which only the variable was the extraction pH (1.0, 1.5, and 2.0) on the basis of our previous studies on diverse pectin sources. The results showed that the yield of pectin (7.4%-19.8%) was strongly influenced by the extraction pH when the other parameters, namely the solid to liquid extractant (S/L) ratio, temperature (T °C), and time (t) were fixed to 1:25 (w/v), 75°C, and 90 min, respectively. Likewise, the galacturonic acid content (GalA: 61.4%-79.2%), total neutral sugar content (TNS: 9.1%-22.5%), degree of branching (3.5%-13.9%), homogalacturonan (HG) to rhamnogalacturonan-I (RG-I) ratio (2.2-5.6), degree of methylesterification (DM: 54-77), viscosity average molecular weight (Mν: 57-82), and gelling capacity (GC: 124-158) were all affected by the extraction pH. The optimum pH for producing pectin with good yield, quality characteristics (GalA > 65%, DM > 60, Mν > 80 kDa), and gelling capacity (GC > 150), from the peel of P. trifoliata fruit, was found to be pH 1.5.
