Pre-harvest water deficit(PHWD)plays an important role in sugar accumulation of citrus fruit.However,the mechanism is not known well.Here,it was confirmed that PHWD promoted sucrose accumulation of citrus fruit,but ha...Pre-harvest water deficit(PHWD)plays an important role in sugar accumulation of citrus fruit.However,the mechanism is not known well.Here,it was confirmed that PHWD promoted sucrose accumulation of citrus fruit,but had limited effect on fructose,glucose and total acid.A sucrose transporter,Cs SUT1,which localizes to the plasma membrane,was demonstrated to function in sucrose transport induced by PHWD.Compared to wild-type,Cs SUT1 overexpression in citrus calli stimulated sucrose,fructose and glucose accumulation,while its silencing in juice sacs reduced sucrose accumulation.Increased sugar accumulation in transgenic lines enhanced plant drought tolerance,and resulted in decreased electrolyte leakage,malondialdehyde and hydrogen peroxide contents,as well as increased superoxide dismutase activity and proline contents.An abscisic acid(ABA)-responsive transcription factor,Cs ABF3,was found with a same expression pattern with Cs SUT1 under PHWD.Yeast one-hybrid,electrophoretic mobility shift assay and dual-luciferase assays all revealed that Cs ABF3 directly bound with the Cs SUT1 promoter by ABA responsive elements.When Cs ABF3 was overexpressed in citrus calli,the sucrose,fructose and glucose concentration increased correspondingly.Further,transgenic studies demonstrated that Cs ABF3 could affect sucrose accumulation by regulating Cs SUT1.Overall,this study revealed a regulation of Cs ABF3 promoting Cs SUT1 expression and sucrose accumulation in response to PHWD.Our results provide a detail insight into the quality formation of citrus fruit.展开更多
Citrus is the typical mycorrhizal fruit tree species establishing symbiosis with arbuscular mycorrhizal (AM) fungi. However, arbuscule development and senescence in colonized citrus roots, especially in response to dr...Citrus is the typical mycorrhizal fruit tree species establishing symbiosis with arbuscular mycorrhizal (AM) fungi. However, arbuscule development and senescence in colonized citrus roots, especially in response to drought stress, remain unclear, which is mainly due to the difficulty in clearing and staining lignified roots with the conventional method. Here, we improved the observation of colonized roots of citrus plants with the sectioning method, which enabled the clear observation of AM fungal structures. Furthermore, we investigated the effects of one week of drought stress on arbuscule development and senescence with the sectioning method. Microscopy observations indicated that drought stress significantly decreased mycorrhizal colonization (F%and M%) although it did not affect plant growth performance. Fluorescence probes (WGA 488 and/or Nile red) revealed that drought stress inhibited arbuscule development by increasing the percentage of arbuscules at the early stage and decreasing the percentages of arbuscules at the midterm and mature stages. Meanwhile, drought stress accelerated arbuscule senescence, which was characterized by the increased accumulation of neutral lipids. Overall, the sectioning method developed in this study enables the in-depth investigation of arbuscule status, and drought stress can inhibit arbuscule development but accelerate arbuscule senescence in the colonized roots of citrus plants. This study paves the way to elaborately dissecting the arbuscule dynamics in the roots of fruit tree species in response to diverse abiotic stresses.展开更多
One of the main diseases that adversely impacts the global citrus industry is citrus bacterial canker(CBC),caused by the bacteria Xanthomonas citri subsp.citri(Xcc).Response to CBC is a complex process,with both prote...One of the main diseases that adversely impacts the global citrus industry is citrus bacterial canker(CBC),caused by the bacteria Xanthomonas citri subsp.citri(Xcc).Response to CBC is a complex process,with both proteinDNA as well as protein–protein interactions for the regulatory network.To detect such interactions in CBC resistant regulation,a citrus high-throughput screening system with 203 CBC-inducible transcription factors(TFs),were developed.Screening the upstream regulators of target by yeast-one hybrid(Y1H)methods was also performed.A regulatory module of CBC resistance was identified based on this system.One TF(CsDOF5.8)was explored due to its interactions with the 1-kb promoter fragment of CsPrx25,a resistant gene of CBC involved in reactive oxygen species(ROS)homeostasis regulation.Electrophoretic mobility shift assay(EMSA),dual-LUC assays,as well as transient overexpression of CsDOF5.8,further validated the interactions and transcriptional regulation.The CsDOF5.8–CsPrx25 promoter interaction revealed a complex pathway that governs the regulation of CBC resistance via H2O2homeostasis.The high-throughput Y1H/Y2H screening system could be an efficient tool for studying regulatory pathways or network of CBC resistance regulation.In addition,it could highlight the potential of these candidate genes as targets for efforts to breed CBC-resistant citrus varieties.展开更多
Huanglongbing(HLB) is a devastating disease that has led to an acute crisis for growers of citrus, one of the world's most important fruit crops. The phloem-feeding Asian citrus psyllid(ACP), Diaphorina citri, is ...Huanglongbing(HLB) is a devastating disease that has led to an acute crisis for growers of citrus, one of the world's most important fruit crops. The phloem-feeding Asian citrus psyllid(ACP), Diaphorina citri, is the main pest at the new shoot stage and is the only natural vector of HLB pathogenic bacteria. Little is known about how plants perceive and defend themselves from this destructive pest. Here, we characterized changes in the expression of various genes in citrus plants that were continuously infested by D. citri for different durations(12, 24, and 48 h). A total of 5 219 differentially expressed genes(DEGs) and 643 common DEGs were identified across all time points. Several pathways related to defense were activated, such as peroxisome, alpha-linolenic acid metabolism, and phenylpropanoid and terpenoid biosynthesis, and some pathways related to growth and signal transduction were suppressed in response to D. citri infestation. The expression of genes including kinases(CML44, CIPK6, and XTH6), phytohormones(SAMT, LOX6, and NPR3), transcription factors(bHLH162, WRKY70, and WRKY40), and secondary metabolite synthesis-related genes(PAL, 4CL2, UGT74B1 and CYP82G1) was significantly altered in response to D. citri infestation. The findings of this study greatly enhance our understanding of the mechanisms underlying the defense response of citrus plants to D. citri infestation at the molecular level. Functional characterization of the candidate defense-related genes identified in this study will aid the molecular breeding of insect-resistant citrus varieties.展开更多
Cytochrome P450s(CYPs)are the largest family of enzymes in plant and play multifarious roles in development and defense but the available information about the CYP superfamily in citrus is very limited.Here we provide...Cytochrome P450s(CYPs)are the largest family of enzymes in plant and play multifarious roles in development and defense but the available information about the CYP superfamily in citrus is very limited.Here we provide a comprehensive genome-wide analysis of the CYP superfamily in Citrus clementina genome,identifying 301 CYP genes grouped into ten clans and 49 families.The characteristics of both gene structures and motif compositions strongly supported the reliability of the phylogenetic relationship.Duplication analysis indicated that tandem duplication was the major driving force of expansion for this superfamily.Promoter analysis revealed numerous cis-acting elements related to various responsiveness.RNA-seq data elucidated their expression patterns in citrus fruit peel both during development and in response to UV-B.Furthermore,we characterize a UV-B-induced CYP gene(Ciclev10019637m,designated CitF3′H)as a f lavonoid 3-hydroxylase for the first time.CitF3 H catalyzed numerous f lavonoids and favored naringenin in yeast assays.Virusinduced silencing of CitF3′ H in citrus seedlings significantly reduced the levels of 3′-hydroxylated f lavonoids and their derivatives.These results together with the endoplasmic reticulum-localization of CitF3 H in plant suggest that this enzyme is responsible for the biosynthesis of 3-hydroxylated f lavonoids in citrus.Taken together,our findings provide extensive information about the CYP superfamily in citrus and contribute to further functional verification.展开更多
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
Protoplast has been widely used in biotechnologies to circumvent the breeding obstacles in citrus, including long juvenility, polyembryony, and male/female sterility. The protoplast-based transient gene expression sys...Protoplast has been widely used in biotechnologies to circumvent the breeding obstacles in citrus, including long juvenility, polyembryony, and male/female sterility. The protoplast-based transient gene expression system is a powerful tool for gene functional characterization and CRISPR/Cas9 genome editing in higher plants, but it has not been widely used in citrus. In this study, the polyethylene glycol(PEG)-mediated method was optimized for citrus callus protoplast transfection, with an improved transfection efficiency of 68.4%. Consequently, the efficiency of protein subcellular localization assay was increased to 65.8%, through transient expression of the target gene in protoplasts that stably express the fluorescent organelle marker protein. The gene editing frequencies in citrus callus protoplasts reached 14.2% after transient expression of CRISPR/Cas9 constructs. We demonstrated that the intronic polycistronic tRNAgRNA(inPTG) genome editing construct was functional in both the protoplast transient expression system and epicotyl stable transformation system in citrus. With this optimized protoplast transient expression system, we improved the efficiency of protein subcellular localization assay and developed the genome editing system in callus protoplasts, which provides an approach for prompt test of CRISPR vectors.展开更多
Citrus fruit crops are among the world’s most important agricultural products,but pests and diseases impact their cultivation,resulting in yield and quality losses.Computer vision and machine learning have been widel...Citrus fruit crops are among the world’s most important agricultural products,but pests and diseases impact their cultivation,resulting in yield and quality losses.Computer vision and machine learning have been widely used to detect and classify plant diseases over the last decade,allowing for early disease detection and improving agricultural production.This paper presented an automatic system for the early detection and classification of citrus plant diseases based on a deep learning(DL)model,which improved accuracy while decreasing computational complexity.The most recent transfer learning-based models were applied to the Citrus Plant Dataset to improve classification accuracy.Using transfer learning,this study successfully proposed a Convolutional Neural Network(CNN)-based pre-trained model(EfficientNetB3,ResNet50,MobiNetV2,and InceptionV3)for the identification and categorization of citrus plant diseases.To evaluate the architecture’s performance,this study discovered that transferring an EfficientNetb3 model resulted in the highest training,validating,and testing accuracies,which were 99.43%,99.48%,and 99.58%,respectively.In identifying and categorizing citrus plant diseases,the proposed CNN model outperforms other cuttingedge CNN model architectures developed previously in the literature.展开更多
Sexual reproduction in plants is the main pathway for creating new genetic combinations in modern agriculture.In heterozygous plants,after the identification of a plant with desired traits,vegetative propagation(cloni...Sexual reproduction in plants is the main pathway for creating new genetic combinations in modern agriculture.In heterozygous plants,after the identification of a plant with desired traits,vegetative propagation(cloning)is the primary path to create genetically uniform plants.Another natural plant mechanism that creates genetically uniform plants(clones)is apomixis.In fruit crops like citrus and mango,sporophytic apomixis results in polyembryony,where seeds contain multiple embryos,one of which is sexually originated and the others are vegetative clones of the parent mother tree.Utilizing the mango genome and genetic analysis of a diverse germplasm collection,we identified MiRWP as the gene that causes polyembryony in mango.There is a strong correlation between a specific insertion in the gene’s promoter region and altered expression in flowers and developing fruitlets,inducing multiple embryos.The MiRWP gene is an ortholog of CitRWP that causes polyembryony in citrus.Based on the data,we speculate that promoter insertion events,which occurred independently in citrus and mango,induced nucellar embryogenesis.The results suggest convergent evolution of polyembryony in the two species.Further work is required to demonstrate the utility of these genes(mango and citrus)in other biological systems as a tool for the clonal production of other crops.展开更多
Cytoplasmic male sterility(CMS)has long been used to produce seedless fruits in perennial woody crops like citrus.A male-sterile somatic cybrid citrus(G1+HBP)was generated by protoplast fusion between a CMS callus par...Cytoplasmic male sterility(CMS)has long been used to produce seedless fruits in perennial woody crops like citrus.A male-sterile somatic cybrid citrus(G1+HBP)was generated by protoplast fusion between a CMS callus parent‘Guoqing No.1’Satsuma mandarin(Citrus unshiu,G1)and a fertile mesophyll parent Hirado Buntan pummelo(Citrus grandis,HBP).To uncover the male-sterile mechanism of G1+HBP,we compared the transcriptome profiles of stamen organ and cell types at five stages between G1+HBP and HBP,including the initial stamen primordia,enlarged stamen primordia,pollen mother cells,tetrads,and microspores captured by laser microdissection.The stamen organ and cell types showed distinct gene expression profiles.A majority of genes involved in stamen development were differentially expressed,especially CgAP3.2,which was downregulated in enlarged stamen primordia and upregulated in tetrads of G1+HBP compared with HBP.Jasmonic acid-and auxin-related biological processes were enriched among the differentially expressed genes of stamen primordia,and the content of jasmonic acid biosynthesis metabolites was higher in flower buds and anthers of G1+HBP.In contrast,the content of auxin biosynthesis metabolites was lower in G1+HBP.The mitochondrial tricarboxylic acid cycle and oxidative phosphorylation processes were enriched among the differentially expressed genes in stamen primordia,meiocytes,and microspores,indicating the dysfunction of mitochondria in stamen organ and cell types of G1+HBP.Taken together,the results indicate that malfunction of mitochondria-nuclear interaction might cause disorder in stamen development,and thus lead to male sterility in the citrus cybrid.展开更多
As the bacterial etiologic agent causing citrus bacterial canker(CBC),Xanthomonas citri subsp.citri(Xcc)seriously impacts citrus plantation and fruit production globally.In an earlier study,we demonstrated that CsBZIP...As the bacterial etiologic agent causing citrus bacterial canker(CBC),Xanthomonas citri subsp.citri(Xcc)seriously impacts citrus plantation and fruit production globally.In an earlier study,we demonstrated that CsBZIP40 can positively impact CBC resistance in the sweet orange(Citrus sinensis).However,the mechanistic basis for the protective benefits conferred by CsBZIP40 is yet to be delineated.Here,we show that CsBZIP40 positively regulates CBC resistance and reactive oxygen species(ROS)homeostasis in transgenic sweet orange overexpressing CsBZIP40.CsBZIP40 directly binds to the TGA-box of the CsWRKY43 promoter to repress its transcriptional activity.CsWRKY43 overexpression induces CBC susceptibility in transgenic sweet oranges.In contrast,its inhibition produces strong resistance to CBC.CsWRKY43 directly binds to the W-boxes of the CsPrx53 and CsSOD13 promoters to positively regulate the activities of these antioxidant enzymes,resulting in the negative regulation of ROS homeostasis and CBC resistance in sweet orange plants.CsPrx53/CsSOD13 knockdown enhances ROS accumulation and CBC resistance.Overall,our results outline a regulatory pathway through which CsBZIP40 transcriptionally represses CsWRKY43-CsPrx53/CsSOD13 cascade-mediated ROS scavenging in a manner conducive to CBC resistance.These mechanisms underscore the potential importance of CsBZIP40,CsWRKY43,CsPrx53,and CsSOD13,providing promising strategies for the prevention of CBC.展开更多
The most damaging citrus diseases are Huanglongbing(HLB)and citrus canker,which are caused by Candidatus Liberibacter asiaticus(CaLas)and Xanthomonas citri pv.citri(Xcc),respectively.Endolysins from bacteriophages are...The most damaging citrus diseases are Huanglongbing(HLB)and citrus canker,which are caused by Candidatus Liberibacter asiaticus(CaLas)and Xanthomonas citri pv.citri(Xcc),respectively.Endolysins from bacteriophages are a possible option for disease resistance in plant breeding.Here,we report improvement of citrus resistance to HLB and citrus canker using the LasLYS1 and LasLYS2 endolysins from CaLas.LasLYS2 demonstrated bactericidal efficacy against several Rhizobiaceae bacteria and Xcc,according to inhibition zone analyses.The two genes,driven by a strong promoter from Cauliflower mosaic virus,35S,were integrated into Carrizo citrange via Agrobacterium-mediated transformation.More than 2 years of greenhouse testing indicated that LasLYS2 provided substantial and long-lasting resistance to HLB,allowing transgenic plants to retain low CaLas titers and no obvious symptoms while also clearing CaLas from infected plants in the long term.LasLYS2 transgenic plants with improved HLB resistance also showed resistance to Xcc,indicating that LasLYS2 had dual resistance to HLB and citrus canker.A microbiome study of transgenic plants revealed that the endolysins repressed Xanthomonadaceae and Rhizobiaceae populations in roots while increasing Burkholderiaceae and Rhodanobacteraceae populations,which might boost the citrus defense response,according to transcriptome analysis.We also found that Lyz domain 2 is the key bactericidal motif of LasLYS1 and LasLYS2.Four endolysins with potential resistance to HLB and citrus canker were found based on the structures of LasLYS1 and LasLYS2.Overall,the work shed light on the mechanisms of resistance of CaLas-derived endolysins,providing insights for designing endolysins to develop broad-spectrum disease resistance in citrus.展开更多
Citrus Tristeza Virus(CTV),usually occurs in nature as a mixture of genotypes.Six naturally infected citrus(Citrus sinensis)trees grafted on sour orange rootstock were collected from three citrus growing governorates ...Citrus Tristeza Virus(CTV),usually occurs in nature as a mixture of genotypes.Six naturally infected citrus(Citrus sinensis)trees grafted on sour orange rootstock were collected from three citrus growing governorates in Egypt(Sharqia,Qalyubia and Garbia).In this study,RT-PCR,Single-Strand Conformation Polymorphism(SSCP)and nucleotide sequence analysis were used for four independent CTV genomic regions(p65,p18,p20,and p23)to detect and assess the sequence and genetic variabilities among CTV Egyptian isolates.RTPCR products(650 bp)for the CTV p23 gene obtained from the selected isolates were used for the SSCP analysis and DNA sequencing.SSCP patterns of p23 gene for individual isolates yielded different complex haplotype patterns.Nucleotide sequence analysis of p23 region amplified from six isolates under study revealed that p23 shared high nucleotide identity 98.7%with T36 isolate from USA,Florida.Phylogenetic analysis of p23 gene indicated a close evolutionary relationship between all examined isolates and Qaha isolate(T36 isolate group),suggesting that they may have originated from closely related ancestors.Nucleotide sequence analysis of the three genes located on CTV 3′-coterminal overhang,p18,p20 and p65,amplified from isolate A3,Sharqia governorate,revealed that the p18,p65,and p20 genes were related to the T3-KB isolate from South Africa with 99%–100%sequence homology.Phylogenetic relationship analysis for p65,p18 and p20 ORFs clustered the current A3 isolate with T3 genotype group.The recombination analysis identified three of six isolates from Sharqia,and Garbia as potential recombinant for p23 gene.The isolates T36 and T3 were identified as major donors for recombination events in isolate A3.Our results concluded that p23 ORF likely to be as a hotspot region for recombination and originated through recombination event.The current study indicated that recombination is an important factor for the origin of CTV strains in Egypt.展开更多
Environmental changes derived from global warming and human activities increase the intensity and frequency of stressful conditions for plants.Multiple abiotic factors acting simultaneously enhance stress pressure and...Environmental changes derived from global warming and human activities increase the intensity and frequency of stressful conditions for plants.Multiple abiotic factors acting simultaneously enhance stress pressure and drastically reduce plant growth,yield,and survival.Stress combination causes a specific stress situation that induces a particular plant response different to the sum of responses to the individual stresses.Here,by comparing transcriptomic and proteomic profiles to different abiotic stress combinations in two citrus genotypes,Carrizo citrange(Citrus sinensis×Poncirus trifoliata)and Cleopatra mandarin(Citrus reshni),with contrasting tolerance to different abiotic stresses,we revealed key responses to the triple combination of heat stress,high irradiance and drought.The specific transcriptomic response to this stress combination in Carrizo was directed to regulate RNA metabolic pathways and translation processes,potentially conferring an advantage with respect to Cleopatra.In addition,we found endoplasmic reticulum stress response as common to all individual and combined stress conditions in both genotypes and identified the accumulation of specific groups of heat shock proteins(HSPs),such as small HSPs and HSP70s,and regulators of the unfolded protein response,BiP2 and PDIL2-2,as possible factors involved in citrus tolerance to triple stress combination.Taken together,our findings provide new insights into the acclimation process of citrus plants to multiple stress combination,necessary for increasing crop tolerance to the changing climatic conditions.展开更多
Recent advances in genome sequencing and assembly techniques have made it possible to achieve chromosome level reference genomes for citrus.Relatively few genomes have been anchored at the chromosome level and/or are ...Recent advances in genome sequencing and assembly techniques have made it possible to achieve chromosome level reference genomes for citrus.Relatively few genomes have been anchored at the chromosome level and/or are haplotype phased,with the available genomes of varying accuracy and completeness.We now report a phased high-quality chromosome level genome assembly for an Australian native citrus species;Citrus australis(round lime)using highly accurate PacBio HiFi long reads,complemented with Hi-C scaffolding.Hifiasm with Hi-C integrated assembly resulted in a 331 Mb genome of C.australis with two haplotypes of nine pseudochromosomes with an N50 of 36.3 Mb and 98.8% genome assembly completeness(BUSCO).Repeat analysis showed that more than 50% of the genome contained interspersed repeats.Among them,LTR elements were the predominant type(21.0%),of which LTR Gypsy(9.8%)and LTR copia(7.7%)elements were the most abundant repeats.A total of 29464 genes and 32009 transcripts were identified in the genome.Of these,28222 CDS(25753 genes)had BLAST hits and 21401 CDS(75.8%)were annotated with at least one GO term.Citrus specific genes for antimicrobial peptides,defense,volatile compounds and acidity regulation were identified.The synteny analysis showed conserved regions between the two haplotypes with some structural variations in Chromosomes 2,4,7 and 8.This chromosome scale,and haplotype resolved C.australis genome will facilitate the study of important genes for citrus breeding and will also allow the enhanced definition of the evolutionary relationships between wild and domesticated citrus species.展开更多
Carotenoids directly influence citrus fruit color and nutritional value,which is critical to consumer acceptance.Elucidating the potential molecular mechanism underlying carotenoid metabolism is of great importance fo...Carotenoids directly influence citrus fruit color and nutritional value,which is critical to consumer acceptance.Elucidating the potential molecular mechanism underlying carotenoid metabolism is of great importance for improving fruit quality.Despite the well-established carotenoid biosynthetic pathways,the molecular regulatory mechanism underlying carotenoid metabolism remains poorly understood.Our previous studies have reported that the Myc-type basic helix-loop-helix(bHLH)transcription factor(TF)regulates citrus proanthocyanidin biosynthesis.Transgenic analyses further showed that overexpression of CsTT8 could significantly promote carotenoid accumulation in transgenic citrus calli,but its regulatory mechanism is still unclear.In the present study,we found that overexpression of CsTT8 enhances carotenoid content in citrus fruit and calli by increasing the expression of CsDXR,CsHDS,CsHDR,CsPDS,CsLCYE,CsZEP,and CsNCED2,which was accompanied by changes in the contents of abscisic acid and gibberellin.The in vitro and in vivo assays indicated that CsTT8 directly bound to the promoters of CsDXR,CsHDS,and CsHDR,the keymetabolic enzymes of the methylerythritol 4-phosphate(MEP)pathway,thus providing precursors for carotenoid biosynthesis and transcriptionally activating the expression of these three genes.In addition,CsTT8 activated the promoters of four key carotenoid biosynthesis pathway genes,CsPDS,CsLCYE,CsZEP,and CsNCED2,directly promoting carotenoid biosynthesis.This study reveals a novel network of carotenoid metabolism regulated by CsTT8.Our findings will contribute to manipulating carotenoid metabolic engineering to improve the quality of citrus fruit and other crops.展开更多
Abstract As an essential horticultural crop,Citrus has carotenoid diversity,which affects its aesthetic and nutritional values.β,β-Xanthophylls are the primary carotenoids accumulated in citrus fruits,and non-heme d...Abstract As an essential horticultural crop,Citrus has carotenoid diversity,which affects its aesthetic and nutritional values.β,β-Xanthophylls are the primary carotenoids accumulated in citrus fruits,and non-heme di-iron carotene hydroxylase(BCH)enzymes are mainly responsible forβ,β-xanthophyll synthesis.Previous studies have focused on the hydroxylation of BCH1,but the role of its paralogous gene in citrus,BCH2,remains largely unknown.In this study,we revealed theβ-hydroxylation activity of citrus BCH2(CsBCH2)for the first time through the functional complementation assay using Escherichia coli,although CsBCH2 exhibited a lower activity in hydroxylatingβ-carotene intoβ-cryptoxanthin than citrus BCH1(CsBCH1).Our results showed that overexpression of CsBCH2 in citrus callus increased xanthophyll proportion and plastoglobule size with feedback regulation of carotenogenic gene expression.This study revealed the distinct expression patterns and functional characteristics of two paralogous genes,CsBCH1 and CsBCH2,and illustrated the backup compensatory role of CsBCH2 for CsBCH1 in citrus xanthophyll biosynthesis.The independent function of CsBCH2 and its cooperative function with CsBCH1 inβ-cryptoxanthin biosynthesis suggested the potential of CsBCH2 to be employed for expanding the synthetic biology toolkit in carotenoid engineering。展开更多
[Objectives]The paper was to explore a faster and more accurate detection method for citrus psyllid to prevent and control yellow-shoot disease and inhibit its transmission.[Methods]We used an improved YOLOX based edg...[Objectives]The paper was to explore a faster and more accurate detection method for citrus psyllid to prevent and control yellow-shoot disease and inhibit its transmission.[Methods]We used an improved YOLOX based edge detection method for psyllid,added Convolutional Block Attention Module(CBAM)to the backbone network,and further extracted important features in the channel and space dimensions.The Cross Entropy Loss in the object loss was changed to Focal Loss to further reduce the missed detection rate.[Results]The algorithm described in the study fitted in with the detection platform of psyllid.The data set of psyllid was taken in Lianjiang Orange Garden,Zhanjiang City,Guangdong Province,deeply adapted to the actual needs of agricultural and rural development.Based on YOLOX model,the backbone network and loss function were improved to achieve a more excellent detection method of citrus psyllid.The AP value of 85.66%was obtained on the data set of citrus psyllid,which was 2.70%higher than that of the original model,and the detection accuracies were 8.61%,4.32%and 3.62%higher than that of YOLOv3,YOLOv4-Tiny and YOLOv5-s,respectively,which had been greatly improved.[Conclusions]The improved YOLOX model can better identify citrus psyllid,and the accuracy rate has been improved,laying a foundation for the subsequent real-time detection platform.展开更多
In the present study, Citrus maxima essential crude oil extract was used to prepare combinatorial formulations and workers of Indian white termite Odontotermes obesus were treated topically with 40% and 80% of 24 hr L...In the present study, Citrus maxima essential crude oil extract was used to prepare combinatorial formulations and workers of Indian white termite Odontotermes obesus were treated topically with 40% and 80% of 24 hr LD50 values of these formulations. In subsequent bioassays levels of various enzymes i.e. alkaline phosphatase, acid phosphatase, glutamate oxaloacetate transaminase, glutamate pyruvate transaminase and acetylcholinesterase were evaluated to determine the anti-termite efficacy of Citrus maxima essential oil based combinatorial formulations. S-RET-A, S-RET-B and S-RET-C caused significant (p > 0.05) decrease in glutamate oxaloacetate transaminase i.e. 87.47%, 86.81% and 81.77% & 82.04%, 79.39% and 74.75% respectively at 16 h treatment. In vivo exposure of 40% and 80% of LD<sub>50</sub> of combinatorial formulations caused very significant (p > 0.05) reduction in all the test enzymes i.e. alkaline phosphatase, acid phosphatase, glutamate oxaloacetate transaminase and glutamate pyruvate transaminase expect acetylcholinesterase levels after 16 h all tested treatments in comparison to control. Both dose-response and time period were found important in physiological alteration in levels of various enzymes. Combinatorial mixtures of Citrus essential oils have shown synergistic activity against termites. The research findings of the present study would help termite control in crop fields, gardens and houses in a sustainable way.展开更多
The general objective of the work is to compare the effect of the addition of synthetic citric acid compared to the addition of natural fruit juice of Citrus aurantiifolia on the conservation of drink based on red cha...The general objective of the work is to compare the effect of the addition of synthetic citric acid compared to the addition of natural fruit juice of Citrus aurantiifolia on the conservation of drink based on red chalice H. sabdariffa. The tests were carried out over a period of 5 weeks at 4°C and 37°C with seven batches of beverage samples prepared at the rate of a calyx/water ratio of 1/40 (kg·kg<sup>-1</sup>) and added respectively citric acid at 1, 2 and 4 g·L<sup>-1</sup> and lemon juice at 10, 20 and 40 mL·L<sup>-1</sup>. The characterization of the different batches of beverages was carried out from day one. A follow-up of the residual anthocyanin content and the intensity of the red coloring were carried out over five weeks. The anthocyanin concentration was determined by the pH-differential method. The red color degradation index is determined based on the CIELAB color system (L*, a*, b* and L*). R and Minitab 18 software was used for data processing. The results of the monitoring of the parameters showed that the concentration of 1 g AC L<sup>-1</sup> retains 2.7 mg more of the anthocyanins than adding 10 mL JC L<sup>-1</sup> and longer maintains red color when stored at 4°C and 37°C/5 weeks. The 2 g AC L<sup>-1</sup> and 4 g AC L<sup>-1</sup> ratios have the same effects as the addition of 20 and 40 mL of lemon juice, all accelerating the degradation of anthocyanins and the red color. After 5 weeks of storage at 37°C, the effect of the temperature combined with the increase in the acidity of the samples (2 to 4 g AC L<sup>-1</sup> and 20 to 40 ml JC L<sup>-1</sup>), have accelerated the total disappearance of the red color on all samples.展开更多
基金supported by the National Natural Science Foundation of China(Grant No.32172520)the earmarked fund for China Agriculture Research System(Grant No.CARS-26)。
文摘Pre-harvest water deficit(PHWD)plays an important role in sugar accumulation of citrus fruit.However,the mechanism is not known well.Here,it was confirmed that PHWD promoted sucrose accumulation of citrus fruit,but had limited effect on fructose,glucose and total acid.A sucrose transporter,Cs SUT1,which localizes to the plasma membrane,was demonstrated to function in sucrose transport induced by PHWD.Compared to wild-type,Cs SUT1 overexpression in citrus calli stimulated sucrose,fructose and glucose accumulation,while its silencing in juice sacs reduced sucrose accumulation.Increased sugar accumulation in transgenic lines enhanced plant drought tolerance,and resulted in decreased electrolyte leakage,malondialdehyde and hydrogen peroxide contents,as well as increased superoxide dismutase activity and proline contents.An abscisic acid(ABA)-responsive transcription factor,Cs ABF3,was found with a same expression pattern with Cs SUT1 under PHWD.Yeast one-hybrid,electrophoretic mobility shift assay and dual-luciferase assays all revealed that Cs ABF3 directly bound with the Cs SUT1 promoter by ABA responsive elements.When Cs ABF3 was overexpressed in citrus calli,the sucrose,fructose and glucose concentration increased correspondingly.Further,transgenic studies demonstrated that Cs ABF3 could affect sucrose accumulation by regulating Cs SUT1.Overall,this study revealed a regulation of Cs ABF3 promoting Cs SUT1 expression and sucrose accumulation in response to PHWD.Our results provide a detail insight into the quality formation of citrus fruit.
基金supported by grants from the Natural Science Foundation of China (Grant No.42077040)the open competition program of top ten critical priorities of Agricultural Science and Technology Innovation for the 14th Five-Year Plan of Guangdong Province (Grant Nos.2022SDZG09,2023SDZG09)+1 种基金the Natural Science Foundation of Guangdong (Grant No.2021B1515010868)the GDAS Project of Science and Technology Development(2021GDASYL-20210103023)。
文摘Citrus is the typical mycorrhizal fruit tree species establishing symbiosis with arbuscular mycorrhizal (AM) fungi. However, arbuscule development and senescence in colonized citrus roots, especially in response to drought stress, remain unclear, which is mainly due to the difficulty in clearing and staining lignified roots with the conventional method. Here, we improved the observation of colonized roots of citrus plants with the sectioning method, which enabled the clear observation of AM fungal structures. Furthermore, we investigated the effects of one week of drought stress on arbuscule development and senescence with the sectioning method. Microscopy observations indicated that drought stress significantly decreased mycorrhizal colonization (F%and M%) although it did not affect plant growth performance. Fluorescence probes (WGA 488 and/or Nile red) revealed that drought stress inhibited arbuscule development by increasing the percentage of arbuscules at the early stage and decreasing the percentages of arbuscules at the midterm and mature stages. Meanwhile, drought stress accelerated arbuscule senescence, which was characterized by the increased accumulation of neutral lipids. Overall, the sectioning method developed in this study enables the in-depth investigation of arbuscule status, and drought stress can inhibit arbuscule development but accelerate arbuscule senescence in the colonized roots of citrus plants. This study paves the way to elaborately dissecting the arbuscule dynamics in the roots of fruit tree species in response to diverse abiotic stresses.
基金funded by the National Key Research and Development Program of China(2022YFD1201600)the earmarked fund for the China Agriculture Research System(CARS-26)+1 种基金the Fundamental Research Funds for the Central Universities,China(SWU-XDJH202308)the Science and Technology Research Program of Chongqing Municipal Education Commission,China(KJQN202001418)。
文摘One of the main diseases that adversely impacts the global citrus industry is citrus bacterial canker(CBC),caused by the bacteria Xanthomonas citri subsp.citri(Xcc).Response to CBC is a complex process,with both proteinDNA as well as protein–protein interactions for the regulatory network.To detect such interactions in CBC resistant regulation,a citrus high-throughput screening system with 203 CBC-inducible transcription factors(TFs),were developed.Screening the upstream regulators of target by yeast-one hybrid(Y1H)methods was also performed.A regulatory module of CBC resistance was identified based on this system.One TF(CsDOF5.8)was explored due to its interactions with the 1-kb promoter fragment of CsPrx25,a resistant gene of CBC involved in reactive oxygen species(ROS)homeostasis regulation.Electrophoretic mobility shift assay(EMSA),dual-LUC assays,as well as transient overexpression of CsDOF5.8,further validated the interactions and transcriptional regulation.The CsDOF5.8–CsPrx25 promoter interaction revealed a complex pathway that governs the regulation of CBC resistance via H2O2homeostasis.The high-throughput Y1H/Y2H screening system could be an efficient tool for studying regulatory pathways or network of CBC resistance regulation.In addition,it could highlight the potential of these candidate genes as targets for efforts to breed CBC-resistant citrus varieties.
基金supported by Key Realm R&D Program of Guangdong Province (Grant No. 2020B0202090005)Special Fund for Scientific Innovation Strategy-construction of High Level Academy of Agriculture Science (Grant No. R2020PY-JG002)the President Foundation of Guangdong Academy of Agricultural Sciences (Grant No. 202030)。
文摘Huanglongbing(HLB) is a devastating disease that has led to an acute crisis for growers of citrus, one of the world's most important fruit crops. The phloem-feeding Asian citrus psyllid(ACP), Diaphorina citri, is the main pest at the new shoot stage and is the only natural vector of HLB pathogenic bacteria. Little is known about how plants perceive and defend themselves from this destructive pest. Here, we characterized changes in the expression of various genes in citrus plants that were continuously infested by D. citri for different durations(12, 24, and 48 h). A total of 5 219 differentially expressed genes(DEGs) and 643 common DEGs were identified across all time points. Several pathways related to defense were activated, such as peroxisome, alpha-linolenic acid metabolism, and phenylpropanoid and terpenoid biosynthesis, and some pathways related to growth and signal transduction were suppressed in response to D. citri infestation. The expression of genes including kinases(CML44, CIPK6, and XTH6), phytohormones(SAMT, LOX6, and NPR3), transcription factors(bHLH162, WRKY70, and WRKY40), and secondary metabolite synthesis-related genes(PAL, 4CL2, UGT74B1 and CYP82G1) was significantly altered in response to D. citri infestation. The findings of this study greatly enhance our understanding of the mechanisms underlying the defense response of citrus plants to D. citri infestation at the molecular level. Functional characterization of the candidate defense-related genes identified in this study will aid the molecular breeding of insect-resistant citrus varieties.
基金supported by the China Postdoctoral Science Foundation(2021 M692845 and 2021 M700124)the National Natural Science Foundation of China(32101932 and 32072132)the Fundamental Research Funds for the Central Universities(K20220104 and 226-2022-00215).
文摘Cytochrome P450s(CYPs)are the largest family of enzymes in plant and play multifarious roles in development and defense but the available information about the CYP superfamily in citrus is very limited.Here we provide a comprehensive genome-wide analysis of the CYP superfamily in Citrus clementina genome,identifying 301 CYP genes grouped into ten clans and 49 families.The characteristics of both gene structures and motif compositions strongly supported the reliability of the phylogenetic relationship.Duplication analysis indicated that tandem duplication was the major driving force of expansion for this superfamily.Promoter analysis revealed numerous cis-acting elements related to various responsiveness.RNA-seq data elucidated their expression patterns in citrus fruit peel both during development and in response to UV-B.Furthermore,we characterize a UV-B-induced CYP gene(Ciclev10019637m,designated CitF3′H)as a f lavonoid 3-hydroxylase for the first time.CitF3 H catalyzed numerous f lavonoids and favored naringenin in yeast assays.Virusinduced silencing of CitF3′ H in citrus seedlings significantly reduced the levels of 3′-hydroxylated f lavonoids and their derivatives.These results together with the endoplasmic reticulum-localization of CitF3 H in plant suggest that this enzyme is responsible for the biosynthesis of 3-hydroxylated f lavonoids in citrus.Taken together,our findings provide extensive information about the CYP superfamily in citrus and contribute to further functional verification.
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.
基金supported by the National Natural Science Foundation of ChinaChina (Grant Nos. 31872051, 32072528)the Foundation of Hubei Hongshan Laboratory (Grant No.2021hszd009)。
文摘Protoplast has been widely used in biotechnologies to circumvent the breeding obstacles in citrus, including long juvenility, polyembryony, and male/female sterility. The protoplast-based transient gene expression system is a powerful tool for gene functional characterization and CRISPR/Cas9 genome editing in higher plants, but it has not been widely used in citrus. In this study, the polyethylene glycol(PEG)-mediated method was optimized for citrus callus protoplast transfection, with an improved transfection efficiency of 68.4%. Consequently, the efficiency of protein subcellular localization assay was increased to 65.8%, through transient expression of the target gene in protoplasts that stably express the fluorescent organelle marker protein. The gene editing frequencies in citrus callus protoplasts reached 14.2% after transient expression of CRISPR/Cas9 constructs. We demonstrated that the intronic polycistronic tRNAgRNA(inPTG) genome editing construct was functional in both the protoplast transient expression system and epicotyl stable transformation system in citrus. With this optimized protoplast transient expression system, we improved the efficiency of protein subcellular localization assay and developed the genome editing system in callus protoplasts, which provides an approach for prompt test of CRISPR vectors.
基金supported by the“Human Resources Program in Energy Technology”of the Korea Institute of Energy Technology Evaluation and Planning(KETEP)and granted financial resources from the Ministry of Trade,Industry,and Energy,Republic of Korea(No.20204010600090)The authors extend their appreciation to the Deanship of Scientific Research at King Khalid University for funding this work through Small Groups.Project under grant number(R.G.P.1/257/43).
文摘Citrus fruit crops are among the world’s most important agricultural products,but pests and diseases impact their cultivation,resulting in yield and quality losses.Computer vision and machine learning have been widely used to detect and classify plant diseases over the last decade,allowing for early disease detection and improving agricultural production.This paper presented an automatic system for the early detection and classification of citrus plant diseases based on a deep learning(DL)model,which improved accuracy while decreasing computational complexity.The most recent transfer learning-based models were applied to the Citrus Plant Dataset to improve classification accuracy.Using transfer learning,this study successfully proposed a Convolutional Neural Network(CNN)-based pre-trained model(EfficientNetB3,ResNet50,MobiNetV2,and InceptionV3)for the identification and categorization of citrus plant diseases.To evaluate the architecture’s performance,this study discovered that transferring an EfficientNetb3 model resulted in the highest training,validating,and testing accuracies,which were 99.43%,99.48%,and 99.58%,respectively.In identifying and categorizing citrus plant diseases,the proposed CNN model outperforms other cuttingedge CNN model architectures developed previously in the literature.
基金The research was supported by Research Grant No.IS-5106-18R from BARD,The United States-Israel Binational Agricultural Research and Development Fund(granted to A.S.,D.N.K.,Y.C.,and R.O.)by grants No.203-0859(granted to A.S.and R.O.)No.203-0110(granted to Y.C.)from the Chief Scientist of the Israeli Ministry of Agriculture.D.N.K.was supported by a grant from the USDA National Institute of Food and Agriculture(USDA-NIFA 2018-51181-28375).
文摘Sexual reproduction in plants is the main pathway for creating new genetic combinations in modern agriculture.In heterozygous plants,after the identification of a plant with desired traits,vegetative propagation(cloning)is the primary path to create genetically uniform plants.Another natural plant mechanism that creates genetically uniform plants(clones)is apomixis.In fruit crops like citrus and mango,sporophytic apomixis results in polyembryony,where seeds contain multiple embryos,one of which is sexually originated and the others are vegetative clones of the parent mother tree.Utilizing the mango genome and genetic analysis of a diverse germplasm collection,we identified MiRWP as the gene that causes polyembryony in mango.There is a strong correlation between a specific insertion in the gene’s promoter region and altered expression in flowers and developing fruitlets,inducing multiple embryos.The MiRWP gene is an ortholog of CitRWP that causes polyembryony in citrus.Based on the data,we speculate that promoter insertion events,which occurred independently in citrus and mango,induced nucellar embryogenesis.The results suggest convergent evolution of polyembryony in the two species.Further work is required to demonstrate the utility of these genes(mango and citrus)in other biological systems as a tool for the clonal production of other crops.
基金This research was financially supported by the Ministry of Science and Technology of China(2022YFF1003101)the National Natural Science Foundation of China(31530065,31820103011,32202451)the Foundation of Hubei Hongshan Laboratory(2021hszd009).
文摘Cytoplasmic male sterility(CMS)has long been used to produce seedless fruits in perennial woody crops like citrus.A male-sterile somatic cybrid citrus(G1+HBP)was generated by protoplast fusion between a CMS callus parent‘Guoqing No.1’Satsuma mandarin(Citrus unshiu,G1)and a fertile mesophyll parent Hirado Buntan pummelo(Citrus grandis,HBP).To uncover the male-sterile mechanism of G1+HBP,we compared the transcriptome profiles of stamen organ and cell types at five stages between G1+HBP and HBP,including the initial stamen primordia,enlarged stamen primordia,pollen mother cells,tetrads,and microspores captured by laser microdissection.The stamen organ and cell types showed distinct gene expression profiles.A majority of genes involved in stamen development were differentially expressed,especially CgAP3.2,which was downregulated in enlarged stamen primordia and upregulated in tetrads of G1+HBP compared with HBP.Jasmonic acid-and auxin-related biological processes were enriched among the differentially expressed genes of stamen primordia,and the content of jasmonic acid biosynthesis metabolites was higher in flower buds and anthers of G1+HBP.In contrast,the content of auxin biosynthesis metabolites was lower in G1+HBP.The mitochondrial tricarboxylic acid cycle and oxidative phosphorylation processes were enriched among the differentially expressed genes in stamen primordia,meiocytes,and microspores,indicating the dysfunction of mitochondria in stamen organ and cell types of G1+HBP.Taken together,the results indicate that malfunction of mitochondria-nuclear interaction might cause disorder in stamen development,and thus lead to male sterility in the citrus cybrid.
基金This study was funded by the National Key Research and Development Program of China(2022YFD1201600,2021YFD1600800)National Natural Sciences Foundation of China(32202425)Earmarked Funds for the China Agriculture Research System(CARS-26).
文摘As the bacterial etiologic agent causing citrus bacterial canker(CBC),Xanthomonas citri subsp.citri(Xcc)seriously impacts citrus plantation and fruit production globally.In an earlier study,we demonstrated that CsBZIP40 can positively impact CBC resistance in the sweet orange(Citrus sinensis).However,the mechanistic basis for the protective benefits conferred by CsBZIP40 is yet to be delineated.Here,we show that CsBZIP40 positively regulates CBC resistance and reactive oxygen species(ROS)homeostasis in transgenic sweet orange overexpressing CsBZIP40.CsBZIP40 directly binds to the TGA-box of the CsWRKY43 promoter to repress its transcriptional activity.CsWRKY43 overexpression induces CBC susceptibility in transgenic sweet oranges.In contrast,its inhibition produces strong resistance to CBC.CsWRKY43 directly binds to the W-boxes of the CsPrx53 and CsSOD13 promoters to positively regulate the activities of these antioxidant enzymes,resulting in the negative regulation of ROS homeostasis and CBC resistance in sweet orange plants.CsPrx53/CsSOD13 knockdown enhances ROS accumulation and CBC resistance.Overall,our results outline a regulatory pathway through which CsBZIP40 transcriptionally represses CsWRKY43-CsPrx53/CsSOD13 cascade-mediated ROS scavenging in a manner conducive to CBC resistance.These mechanisms underscore the potential importance of CsBZIP40,CsWRKY43,CsPrx53,and CsSOD13,providing promising strategies for the prevention of CBC.
基金supported by the National Key Research and Development Program of China(2021YFD1400800,2022YFD1400200)the National Natural Sciences Foundation of China(31972393)+1 种基金the Fundamental Research Funds for the Central Universities(SWU-XDJH202308,XDJK2018B016)the Earmarked Fund for China Agriculture Research System(CARS-27).
文摘The most damaging citrus diseases are Huanglongbing(HLB)and citrus canker,which are caused by Candidatus Liberibacter asiaticus(CaLas)and Xanthomonas citri pv.citri(Xcc),respectively.Endolysins from bacteriophages are a possible option for disease resistance in plant breeding.Here,we report improvement of citrus resistance to HLB and citrus canker using the LasLYS1 and LasLYS2 endolysins from CaLas.LasLYS2 demonstrated bactericidal efficacy against several Rhizobiaceae bacteria and Xcc,according to inhibition zone analyses.The two genes,driven by a strong promoter from Cauliflower mosaic virus,35S,were integrated into Carrizo citrange via Agrobacterium-mediated transformation.More than 2 years of greenhouse testing indicated that LasLYS2 provided substantial and long-lasting resistance to HLB,allowing transgenic plants to retain low CaLas titers and no obvious symptoms while also clearing CaLas from infected plants in the long term.LasLYS2 transgenic plants with improved HLB resistance also showed resistance to Xcc,indicating that LasLYS2 had dual resistance to HLB and citrus canker.A microbiome study of transgenic plants revealed that the endolysins repressed Xanthomonadaceae and Rhizobiaceae populations in roots while increasing Burkholderiaceae and Rhodanobacteraceae populations,which might boost the citrus defense response,according to transcriptome analysis.We also found that Lyz domain 2 is the key bactericidal motif of LasLYS1 and LasLYS2.Four endolysins with potential resistance to HLB and citrus canker were found based on the structures of LasLYS1 and LasLYS2.Overall,the work shed light on the mechanisms of resistance of CaLas-derived endolysins,providing insights for designing endolysins to develop broad-spectrum disease resistance in citrus.
基金Authors extend their appreciation to Deanship of Scientific Research,King Faisal University,Saudi Arabia,for supporting this research(GRANT494).
文摘Citrus Tristeza Virus(CTV),usually occurs in nature as a mixture of genotypes.Six naturally infected citrus(Citrus sinensis)trees grafted on sour orange rootstock were collected from three citrus growing governorates in Egypt(Sharqia,Qalyubia and Garbia).In this study,RT-PCR,Single-Strand Conformation Polymorphism(SSCP)and nucleotide sequence analysis were used for four independent CTV genomic regions(p65,p18,p20,and p23)to detect and assess the sequence and genetic variabilities among CTV Egyptian isolates.RTPCR products(650 bp)for the CTV p23 gene obtained from the selected isolates were used for the SSCP analysis and DNA sequencing.SSCP patterns of p23 gene for individual isolates yielded different complex haplotype patterns.Nucleotide sequence analysis of p23 region amplified from six isolates under study revealed that p23 shared high nucleotide identity 98.7%with T36 isolate from USA,Florida.Phylogenetic analysis of p23 gene indicated a close evolutionary relationship between all examined isolates and Qaha isolate(T36 isolate group),suggesting that they may have originated from closely related ancestors.Nucleotide sequence analysis of the three genes located on CTV 3′-coterminal overhang,p18,p20 and p65,amplified from isolate A3,Sharqia governorate,revealed that the p18,p65,and p20 genes were related to the T3-KB isolate from South Africa with 99%–100%sequence homology.Phylogenetic relationship analysis for p65,p18 and p20 ORFs clustered the current A3 isolate with T3 genotype group.The recombination analysis identified three of six isolates from Sharqia,and Garbia as potential recombinant for p23 gene.The isolates T36 and T3 were identified as major donors for recombination events in isolate A3.Our results concluded that p23 ORF likely to be as a hotspot region for recombination and originated through recombination event.The current study indicated that recombination is an important factor for the origin of CTV strains in Egypt.
基金This work was supported by Grants PID2019-104062RB-I00 and TED2021-129795B-I00 funded byMCIN/AEI/10.13039/501100011033by the European Union-NextGenerationEU.Funding was also obtained from Universitat Jaume I(UJI-B2022-18)and Generalitat Valenciana(CIAICO/2021/063)+3 种基金DBwas supported by the European Union-NextGenerationEU and the Ministerio de Universidades(MGS/2021/17)S.I.Z.was supported by MCIN(RYC2020-029967-I)T.R.O.was supported by the Fundação Carlos Chagas Filho FAPERJ(E-26/204.192/2021)Coordenação de Aperfeiçoamento de Pessoal de Nível Superior(CAPES-001).
文摘Environmental changes derived from global warming and human activities increase the intensity and frequency of stressful conditions for plants.Multiple abiotic factors acting simultaneously enhance stress pressure and drastically reduce plant growth,yield,and survival.Stress combination causes a specific stress situation that induces a particular plant response different to the sum of responses to the individual stresses.Here,by comparing transcriptomic and proteomic profiles to different abiotic stress combinations in two citrus genotypes,Carrizo citrange(Citrus sinensis×Poncirus trifoliata)and Cleopatra mandarin(Citrus reshni),with contrasting tolerance to different abiotic stresses,we revealed key responses to the triple combination of heat stress,high irradiance and drought.The specific transcriptomic response to this stress combination in Carrizo was directed to regulate RNA metabolic pathways and translation processes,potentially conferring an advantage with respect to Cleopatra.In addition,we found endoplasmic reticulum stress response as common to all individual and combined stress conditions in both genotypes and identified the accumulation of specific groups of heat shock proteins(HSPs),such as small HSPs and HSP70s,and regulators of the unfolded protein response,BiP2 and PDIL2-2,as possible factors involved in citrus tolerance to triple stress combination.Taken together,our findings provide new insights into the acclimation process of citrus plants to multiple stress combination,necessary for increasing crop tolerance to the changing climatic conditions.
文摘Recent advances in genome sequencing and assembly techniques have made it possible to achieve chromosome level reference genomes for citrus.Relatively few genomes have been anchored at the chromosome level and/or are haplotype phased,with the available genomes of varying accuracy and completeness.We now report a phased high-quality chromosome level genome assembly for an Australian native citrus species;Citrus australis(round lime)using highly accurate PacBio HiFi long reads,complemented with Hi-C scaffolding.Hifiasm with Hi-C integrated assembly resulted in a 331 Mb genome of C.australis with two haplotypes of nine pseudochromosomes with an N50 of 36.3 Mb and 98.8% genome assembly completeness(BUSCO).Repeat analysis showed that more than 50% of the genome contained interspersed repeats.Among them,LTR elements were the predominant type(21.0%),of which LTR Gypsy(9.8%)and LTR copia(7.7%)elements were the most abundant repeats.A total of 29464 genes and 32009 transcripts were identified in the genome.Of these,28222 CDS(25753 genes)had BLAST hits and 21401 CDS(75.8%)were annotated with at least one GO term.Citrus specific genes for antimicrobial peptides,defense,volatile compounds and acidity regulation were identified.The synteny analysis showed conserved regions between the two haplotypes with some structural variations in Chromosomes 2,4,7 and 8.This chromosome scale,and haplotype resolved C.australis genome will facilitate the study of important genes for citrus breeding and will also allow the enhanced definition of the evolutionary relationships between wild and domesticated citrus species.
基金supported by the National Natural Science Foundation of China(No.31930095,32172527)the Modern Agro-industry Technology Research System(CARS-26).
文摘Carotenoids directly influence citrus fruit color and nutritional value,which is critical to consumer acceptance.Elucidating the potential molecular mechanism underlying carotenoid metabolism is of great importance for improving fruit quality.Despite the well-established carotenoid biosynthetic pathways,the molecular regulatory mechanism underlying carotenoid metabolism remains poorly understood.Our previous studies have reported that the Myc-type basic helix-loop-helix(bHLH)transcription factor(TF)regulates citrus proanthocyanidin biosynthesis.Transgenic analyses further showed that overexpression of CsTT8 could significantly promote carotenoid accumulation in transgenic citrus calli,but its regulatory mechanism is still unclear.In the present study,we found that overexpression of CsTT8 enhances carotenoid content in citrus fruit and calli by increasing the expression of CsDXR,CsHDS,CsHDR,CsPDS,CsLCYE,CsZEP,and CsNCED2,which was accompanied by changes in the contents of abscisic acid and gibberellin.The in vitro and in vivo assays indicated that CsTT8 directly bound to the promoters of CsDXR,CsHDS,and CsHDR,the keymetabolic enzymes of the methylerythritol 4-phosphate(MEP)pathway,thus providing precursors for carotenoid biosynthesis and transcriptionally activating the expression of these three genes.In addition,CsTT8 activated the promoters of four key carotenoid biosynthesis pathway genes,CsPDS,CsLCYE,CsZEP,and CsNCED2,directly promoting carotenoid biosynthesis.This study reveals a novel network of carotenoid metabolism regulated by CsTT8.Our findings will contribute to manipulating carotenoid metabolic engineering to improve the quality of citrus fruit and other crops.
基金supported by the National Natural Science Foundation of China(No.31930095 and 32172527)the Modern Agro-industry Technology Research System(CARS-26).
文摘Abstract As an essential horticultural crop,Citrus has carotenoid diversity,which affects its aesthetic and nutritional values.β,β-Xanthophylls are the primary carotenoids accumulated in citrus fruits,and non-heme di-iron carotene hydroxylase(BCH)enzymes are mainly responsible forβ,β-xanthophyll synthesis.Previous studies have focused on the hydroxylation of BCH1,but the role of its paralogous gene in citrus,BCH2,remains largely unknown.In this study,we revealed theβ-hydroxylation activity of citrus BCH2(CsBCH2)for the first time through the functional complementation assay using Escherichia coli,although CsBCH2 exhibited a lower activity in hydroxylatingβ-carotene intoβ-cryptoxanthin than citrus BCH1(CsBCH1).Our results showed that overexpression of CsBCH2 in citrus callus increased xanthophyll proportion and plastoglobule size with feedback regulation of carotenogenic gene expression.This study revealed the distinct expression patterns and functional characteristics of two paralogous genes,CsBCH1 and CsBCH2,and illustrated the backup compensatory role of CsBCH2 for CsBCH1 in citrus xanthophyll biosynthesis.The independent function of CsBCH2 and its cooperative function with CsBCH1 inβ-cryptoxanthin biosynthesis suggested the potential of CsBCH2 to be employed for expanding the synthetic biology toolkit in carotenoid engineering。
基金Supported by Research and Development Program in Key Areas of Guangdong Province(2020B0202090005)Lianjiang Think Tank Enterprise Project"Demonstration of Intelligent Monitoring and Ecological Prevention and Control Technology of Red Orange Yellow-shoot Disease and Psyllid in Lianjiang"。
文摘[Objectives]The paper was to explore a faster and more accurate detection method for citrus psyllid to prevent and control yellow-shoot disease and inhibit its transmission.[Methods]We used an improved YOLOX based edge detection method for psyllid,added Convolutional Block Attention Module(CBAM)to the backbone network,and further extracted important features in the channel and space dimensions.The Cross Entropy Loss in the object loss was changed to Focal Loss to further reduce the missed detection rate.[Results]The algorithm described in the study fitted in with the detection platform of psyllid.The data set of psyllid was taken in Lianjiang Orange Garden,Zhanjiang City,Guangdong Province,deeply adapted to the actual needs of agricultural and rural development.Based on YOLOX model,the backbone network and loss function were improved to achieve a more excellent detection method of citrus psyllid.The AP value of 85.66%was obtained on the data set of citrus psyllid,which was 2.70%higher than that of the original model,and the detection accuracies were 8.61%,4.32%and 3.62%higher than that of YOLOv3,YOLOv4-Tiny and YOLOv5-s,respectively,which had been greatly improved.[Conclusions]The improved YOLOX model can better identify citrus psyllid,and the accuracy rate has been improved,laying a foundation for the subsequent real-time detection platform.
文摘In the present study, Citrus maxima essential crude oil extract was used to prepare combinatorial formulations and workers of Indian white termite Odontotermes obesus were treated topically with 40% and 80% of 24 hr LD50 values of these formulations. In subsequent bioassays levels of various enzymes i.e. alkaline phosphatase, acid phosphatase, glutamate oxaloacetate transaminase, glutamate pyruvate transaminase and acetylcholinesterase were evaluated to determine the anti-termite efficacy of Citrus maxima essential oil based combinatorial formulations. S-RET-A, S-RET-B and S-RET-C caused significant (p > 0.05) decrease in glutamate oxaloacetate transaminase i.e. 87.47%, 86.81% and 81.77% & 82.04%, 79.39% and 74.75% respectively at 16 h treatment. In vivo exposure of 40% and 80% of LD<sub>50</sub> of combinatorial formulations caused very significant (p > 0.05) reduction in all the test enzymes i.e. alkaline phosphatase, acid phosphatase, glutamate oxaloacetate transaminase and glutamate pyruvate transaminase expect acetylcholinesterase levels after 16 h all tested treatments in comparison to control. Both dose-response and time period were found important in physiological alteration in levels of various enzymes. Combinatorial mixtures of Citrus essential oils have shown synergistic activity against termites. The research findings of the present study would help termite control in crop fields, gardens and houses in a sustainable way.
文摘The general objective of the work is to compare the effect of the addition of synthetic citric acid compared to the addition of natural fruit juice of Citrus aurantiifolia on the conservation of drink based on red chalice H. sabdariffa. The tests were carried out over a period of 5 weeks at 4°C and 37°C with seven batches of beverage samples prepared at the rate of a calyx/water ratio of 1/40 (kg·kg<sup>-1</sup>) and added respectively citric acid at 1, 2 and 4 g·L<sup>-1</sup> and lemon juice at 10, 20 and 40 mL·L<sup>-1</sup>. The characterization of the different batches of beverages was carried out from day one. A follow-up of the residual anthocyanin content and the intensity of the red coloring were carried out over five weeks. The anthocyanin concentration was determined by the pH-differential method. The red color degradation index is determined based on the CIELAB color system (L*, a*, b* and L*). R and Minitab 18 software was used for data processing. The results of the monitoring of the parameters showed that the concentration of 1 g AC L<sup>-1</sup> retains 2.7 mg more of the anthocyanins than adding 10 mL JC L<sup>-1</sup> and longer maintains red color when stored at 4°C and 37°C/5 weeks. The 2 g AC L<sup>-1</sup> and 4 g AC L<sup>-1</sup> ratios have the same effects as the addition of 20 and 40 mL of lemon juice, all accelerating the degradation of anthocyanins and the red color. After 5 weeks of storage at 37°C, the effect of the temperature combined with the increase in the acidity of the samples (2 to 4 g AC L<sup>-1</sup> and 20 to 40 ml JC L<sup>-1</sup>), have accelerated the total disappearance of the red color on all samples.
